The Inhibitory T Cell Receptors PD1 and 2B4 Are Differentially Regulated on CD4 and CD8 T Cells in a Mouse Model of Non-alcoholic Steatohepatitis - PubMed (original) (raw)
The Inhibitory T Cell Receptors PD1 and 2B4 Are Differentially Regulated on CD4 and CD8 T Cells in a Mouse Model of Non-alcoholic Steatohepatitis
Cordula Hansel et al. Front Pharmacol. 2019.
Abstract
Infiltrating CD4 and CD8 T cells have been shown to worsen inflammatory liver damage in non-alcoholic steatohepatitis (NASH). Inhibitory T cell receptors such as the programmed cell death protein 1 (PD1) and the natural killer cell receptor 2B4 regulate the activity of CD4 and CD8 T cells and therefore play an important role in immune tolerance required in the liver. In this study, we investigated the expression profile of inhibitory T cell receptors on CD4 and CD8 T cells in a mouse model of NASH. Male B57BL/6J mice were fed a Western diet for 24 weeks. The expression levels of inhibitory receptors on the surface of intrahepatic and peripheral T cells were measured and correlated with markers of activation (CD107a, CD69, and CD44), metabolic disorder (serum triglycerides, serum cholesterol, γ-glutamyl transferase, hepatic triglycerides), inflammation (serum alanine aminotransferase and aspartate aminotransferase) and hepatic fibrosis (collagen 1A1, α-smooth muscle actin, hydroxyproline). Under Western diet, PD1 is exclusively upregulated on intrahepatic and peripheral CD8+ T cells, whereas the expression level on CD4 T cells is unaffected. In contrast, 2B4 is upregulated liver-specifically on both CD4 and CD8 T cells and unchanged on peripheral T cells. Upregulation of PD1 on CD8 T cells is restricted to CD8 effector memory T cells and correlates with lower levels of degranulation. Similarly, the inhibitory function of PD1 on intrahepatic CD4 T cells is shown by a lower CD69 and CD44 expression on PD1-positive CD4 T cells. In murine steatohepatitis, the upregulation of PD1 on CD8 T cells and 2B4 on CD4 and CD8 T cells potentially limits T cell-mediated liver damage. Therefore, these inhibitory T cell receptors could serve as promising targets of immune-modulatory NASH therapy.
Keywords: NASH; Western diet; inhibitory T cell receptors; liver; natural killer cell receptor 2B4; non-alcoholic steatohepatitis; programmed cell death protein 1.
Figures
FIGURE 1
Metabolic phenotype of mice after feeding a WD for 24 weeks. (A) Body weight curve of male wild-type BL/6J mice fed SC or WD for 24 weeks. (n = 5, ∗∗p < 0.01 and ∗∗∗p < 0.001). (B) Body weight, liver weight, weight of epididymal white adipose tissue and liver to body ratio after feeding SC or WD for 24 weeks. (n = 5), (∗p < 0.05 and ∗∗p < 0.01) (C) Macroscopic aspects of livers from SC and WD fed mice (D) Representative H&E and Oil red O stained liver sections. (E) Intrahepatic triglyceride content after 24 weeks of SC and WD feeding. (n = 5, ∗∗∗p < 0.001). (F) Serum cholesterol and triglyceride levels after 24 weeks of SC or WD feeding. (n = 5, ∗p < 0.05 and ∗∗p < 0.01).
FIGURE 2
Stronger pro-inflammatory immune response, fibrosis development and diabetic phenotype in WD fed mice. (A) ALT and AST serum levels in mice fed a SC or WD for 24 weeks. (n = 5, ∗∗∗p < 0.001) (B) Hepatic gene expression of pro-inflammatory cytokines (tumor necrosis factor α, chemokine ligand 2, interleukin-2 and granzyme B) in mice fed SC or WD for 24 weeks. Values are expressed as relative fold induction over the mean values obtained for SC-fed animals. (n = 5, ∗p < 0.05 and ∗∗∗p < 0.001) (C) Sirius Red-stained liver sections and intrahepatic hydroxyproline levels of SC and WD fed mice. (n = 5, ∗p < 0.05) (D) Western blot analysis with quantitative evaluation for α-SMA in whole liver homogenates of SC and WD fed mice. (n = 4, ∗p < 0.05) (E) mRNA expression levels of α-SMA and Col1A1. Whole liver homogenates of chow and WD fed mice were analyzed via real-time PCR. The quantification is expressed as relative fold induction over the mean values obtained for SC fed mice. (n = 5, ∗p < 0.05 and ∗∗p < 0.01) (F) Mice fed SC or WD for 24 weeks showed an impaired glucose tolerance by stronger increase of blood glucose after intraperitoneal glucose administration compared to equally treated chow diet fed mice. (n = 5, ∗p < 0.05).
FIGURE 3
T cell characterization by flow cytometry in the blood and livers of chow and WD fed mice. (A) T cells (CD45+Hoechst-CD3+NK1.1-) in the blood and liver after SC feeding. (n = 5, ∗∗p < 0.01) (B) Basic PD1 and 2B4 expression in blood and liver CD45+CD4+ and CD45+CD8+ T cells under SC. (n = 5, ∗∗p < 0.01) (C) Absolute leukocyte cell numbers in blood and liver after SC and WD feeding. (n = 5). (D) Absolute T cell numbers in blood and liver after feeding a SC or WD for 24 weeks. (n = 5, ∗p < 0.05) (E) Absolute CD4+ and CD8+ T cell numbers after SC feeding and after a metabolic challenge by a 24 week fed WD. (n = 5, ∗p < 0.05 and ∗∗p < 0.01) (F) Percentage of CD107a positive CD8+ T cells in blood and liver tissue after administration of a SC or WD for 24 weeks. (n = 5, ∗∗∗p < 0.001).
FIGURE 4
Expression of inhibitory T cell receptors PD1 and 2B4 increases on hepatic T cells under WD. (A) Blood and intrahepatic PD1 expressing CD8+ cells were analyzed by Flow Cytometry after SC and WD feeding for 24 weeks. (n = 5, ∗p < 0.05 and ∗∗∗p < 0.001). (B) Percentage of intrahepatic degranulating CD107a+ CD8 T cells of PD1+ and PD1- cells after SC or WD treatment for 24 weeks. (n = 5, ∗p < 0.05). (C) Upregulation of PD1 on intrahepatic CD8+ CD62L+ (Naïve + central memory CD8+ T cells) and CD8+ CD62L- (effector memory CD8+ T cells) T cells after SC or WD. (n = 5, ∗p < 0.05). (D) 2B4 expression on CD8+ T cells in blood and liver after SC or WD feeding for 24 weeks. (n = 5, ∗p < 0.05). (E) PD1 expression on blood and hepatic CD4+ T cells under SC or WD for 24 weeks. (n = 5). (F) Inhibitory receptor 2B4 expression blood and intrahepatic CD4 T cells under SC or WD. (n = 5, ∗p < 0.05).
FIGURE 5
Activation markers CD69 and CD44 are increased under metabolic stress on hepatic CD4 T cells, but not CD8 T cells, while this activation is inhibited in PD1+ CD4 T cells. (A) CD25 expression on blood and liver CD8 T cells under SC or WD for 24 weeks. (n = 5). (B) CD69 expression on blood and on hepatic CD8 T cells after 24 weeks treatment with SC or WD. (n = 5), (∗∗∗p < 0.001). (C) CD69 expression on blood and hepatic CD4 T cells after 24 weeks SC and WD feeding. (n = 5, ∗p < 0.05). (D) CD44 expression on blood and intrahepatic CD4 T cells under SC or WD treatment for 24 weeks. (n = 5, ∗∗p < 0.01 and ∗∗∗p < 0.001). (E) CD25 expression on blood and hepatic CD4 T cells under SC or WD. (n = 5), (∗p < 0.05 and ∗∗∗p < 0.001). (F) CD44 and CD69 expression on PD1+ or PD1- CD4 T cells after SC or WD feeding. (n = 5), (∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001).
FIGURE 6
PD1+ and 2B4+ infiltrating cells are increased in NASH patients. (A) Intrahepatic PD1+ CD4 or CD8+ T cells and (B) 2B4+ CD4 or CD8+ T cells were analyzed by flow cytometry. Analysis include 5 control and 5 NASH patients. Intrahepatic cells were gated FSC/SSC, duplets were excluded, living cells, CD3+, PD1+/CD4+, PD1+/CD8+, 2B4+/CD4+, 2B4+/CD8+. Displayed is the percentage of recorded cells in liver biopsy samples. (C) Intrahepatic PD1+/CD14+/CD68+ and (D) 2B4+/CD14+/CD68+ monocytes were analyzed by flow cytometry. Analysis include 5 control and 5 NASH patients. Intrahepatic cells were gated FSC/SSC, duplets were excluded, living cells, CD14+/CD68+, PD1+ or 2B4+. Displayed is the percentage of recorded cells in liver biopsy samples. (∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001).
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