<i>LAG-3</i> Knockout Exacerbates Immune Liver Injury

doi:10.13523/j.cb.2304017

China Biotechnology

2023, Vol. 43

Issue (8): 38-51 DOI: 10.13523/j.cb.2304017

LAG-3 Knockout Exacerbates Immune Liver Injury

WAN Yue¹,ZHOU Bin²,GAO Rui²,CHEN Hui²,GAO Miao-miao²,WANG Xiao-fang³,DONG Dian-dian³,XIANG Shen-si²,YANG Xiao-ming^1,^2,^**(

),YU Miao^1,^2,^3,^**(

)

1 School of Basic Medicine, Anhui Medical University, Hefei 230032, China
2 Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China
3 School of Life Sciences, Hebei University, Baoding 071002, China

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Abstract

Objective: To investigate the function and mechanism of lymphocyte activation gene 3 (LAG-3) in immune induced liver injury. Methods: Acute immune liver injury model in mice was induced by tail vein injection of concanavalin A (Con A). LAG-3 expression in hepatic immune cells was determined by flow cytometry and real-time PCR. LAG-3 knockout mice were utilized to study the effect of LAG-3 on Con A-induced immune liver injury. Peripheral blood was collected at different time points after Con A injection, and the serum levels of ALT, AST, and cytokines were detected. Liver tissue samples were collected for hematoxylin eosin staining (H & E, hematoxylin eosin staining) and the necrotic area of liver tissue was counted. Intrahepatic immune cell subsets and T cell activation were determined by flow cytometry. The expression of inflammation related genes was detected by real-time quantitative PCR. Yeast two-hybrid screening was performed to find LAG-3 interacting proteins. Results: Con A induced an increase in LAG-3 expression on hepatic immune cells; LAG-3 knockout exacerbated Con A-induced elevation of serum transaminase levels, increased necrotic area of the liver, and delayed resolution of inflammation in mice; LAG-3 knockout did not affect Con A-induced intrahepatic immune cell infiltration; LAG-3 knockout suppressed regulatory T cell expansion and IL-10 expression; MHCII accessory molecule CD74 interacted with LAG-3. Conclusions: LAG-3 knockout aggravates immune liver injury.

Key words： Immune liver injury Lymphocyte activation gene-3 Regulatory T cells Immunosuppression

Received: 10 April 2023 Published: 05 September 2023

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	Yue WAN
	Bin ZHOU
	Rui GAO
	Hui CHEN
	Miao-miao GAO
	Xiao-fang WANG
	Dian-dian DONG
	Shen-si XIANG
	Xiao-ming YANG
	Miao YU

Cite this article:

WAN Yue, ZHOU Bin, GAO Rui, CHEN Hui, GAO Miao-miao, WANG Xiao-fang, DONG Dian-dian, XIANG Shen-si, YANG Xiao-ming, YU Miao. LAG-3 Knockout Exacerbates Immune Liver Injury. China Biotechnology, 2023, 43(8): 38-51.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2304017 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I8/38

Table 1 Primer sequences for real-time PCR

Fig.1 LAG-3^+/+ mouse Con A model with increased LAG-3 expression on the surface of liver lymphocytes (a)Labeling strategy for detecting LAG-3 expression by flow cytometry (b)Flow cytometry results and statistical graphs show CD45⁺ cell count increase after LAG-3⁺^/⁺ mice Con A injection and CD45⁺LAG3⁺ cell count increase after LAG-3⁺^/⁺ mice Con A injection (c)Fluorescence intensity peak plot and statistical plot show an increase in fluorescence intensity in LAG-3⁺^/⁺ mouse cells after injection of Con A (d)qPCR analysis the expression of LAG-3 on the surface of lymphocytes. Data are expressed as the mean ± standard deviation of three independent experiments or representatives of three independent experiments. Unpaired t-test was used to assess that differences were statistically significant

Fig.2 LAG-3 knockdown exacerbates Con A-induced liver injury in mice (a) Gene sequencing results of LAG-3^-^/^- mice (b)Flow cytometry analysis of labeling strategies for the streaming detection of LAG-3 (c)Flow cytometry assay of LAG-3 expression levels in activated primary T cells (d)Transaminase levels in mice at different time points after Con A injection. Data are expressed as the mean ± standard deviation of three independent experiments or representatives of three independent experiments. Unpaired t-test was used to assess that differences were statistically significant

Fig.3 Increased area of liver necrosis in the Con A model of LAG-3 knockout mice (a)HE staining of liver at different time points after tail vein injection in Con A LAG-3 knockdown mice (b)HE staining necrotic area statistics. Data are expressed as the mean ± standard deviation of three independent experiments or representatives of three independent experiments. Unpaired t-test was used to assess that differences were statistically significant

Fig.4 LAG-3 knockdown does not affect Con A-induced intrahepatic immune cell infiltration (a)Flow cytometry antibody labelling strategy for liver immune cells (b)Liver counts of various ne cells at dtypes of immuifferent time points of Con A injections. Data are expressed as the mean ± standard deviation of three independent experiments or representatives of three independent experiments. Unpaired t-test was used to assess that differences were not statistically significant

Fig.5 LAG-3 knockout inhibits regulatory T cell amplification Flow cytometry analysis of antigen-induced regulatory T cell expansion. Data are expressed as the mean ± standard deviation of three independent experiments or representatives of three independent experiments. Unpaired t-test was used to assess that differences were statistically significant

Fig.6 LAG-3 knockdown Con A-induced expression of serum inflammatory cytokines Flow cytometry analysis of serum cell cytokine expression. Data are expressed as the mean ± standard deviation of three independent experiments or representatives of three independent experiments. Unpaired t-test was used to assess that differences were statistically significant

Fig.7 LAG-3 knockdown Con A-induced expression of hepatic inflammatory cytokines RT-PCR analysis of mRNA levels of cytokines in hepatocytes.Data are expressed as the mean ± standard deviation of three independent experiments or representatives of three independent experiments. Unpaired t-test was used to assess that differences were statistically significant

Fig.8 Yeast two-hybrid screening of LAG-3 interacting proteins (a)Test for autonomous activation (b)Positive clone identification chart (c)Rotation verification chart

Table 2 Candidate LAG-3 interacting proteins for yeast two-hybrid screening


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