Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis - PubMed (original) (raw)

. 2021 Dec;75(6):1271-1283.

doi: 10.1016/j.jhep.2021.07.032. Epub 2021 Aug 4.

Hongji Zhang 2, Yu Wang 3, Zachary J Brown 2, Yujia Xia 1, Zheng Huang 4, Chengli Shen 2, Zhiwei Hu 2, Joal Beane 2, Ephraim A Ansa-Addo 5, Hai Huang 2, Dean Tian 6, Allan Tsung 7

Affiliations

Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis

Han Wang et al. J Hepatol. 2021 Dec.

Abstract

Background & aims: Regulatory T-cells (Tregs) impair cancer immunosurveillance by creating an immunosuppressive environment that fosters tumor cell survival. Our previous findings demonstrated that neutrophil extracellular traps (NETs), which are involved both in innate and adaptive immunity, are abundant in livers affected by non-alcoholic steatohepatitis (NASH). However, how NETs interact with Tregs in the development of NASH-associated hepatocellular carcinoma (NASH-HCC) is not known.

Methods: A choline-deficient, high-fat diet+diethylnitrosamine mouse model and the stelic animal model were utilized for NASH-HCC and a western diet mouse model was used for NASH development. Treg depletion was achieved using FoxP3-DTR mice. RNA sequencing was used to explore the mechanism by which NETs could regulate Treg differentiation. Bioenergetic analyses of naïve CD4+ T-cells were assessed by Seahorse.

Results: Although the absolute number of CD4+ T-cells is lower in NASH livers, the Treg subpopulation is selectively increased. Depleting Tregs dramatically inhibits HCC initiation and progression in NASH. There is a positive correlation between increased NET and hepatic Treg levels. RNA sequencing data reveals that NETs impact gene expression profiles in naïve CD4+ T-cells, with the most differentially expressed genes being those involved in mitochondrial oxidative phosphorylation. By facilitating mitochondrial respiration, NETs can promote Treg differentiation. Metabolic reprogramming of naïve CD4+ T-cells by NETs requires toll-like receptor 4. Blockade of NETs in vivo using Pad4-/- mice or DNase I treatment reduces the activity of Tregs.

Conclusions: Tregs can suppress immunosurveillance in the premalignant stages of NASH. NETs facilitate the crosstalk between innate and adaptive immunity in NASH by promoting Treg activity through metabolic reprogramming. Therapies targeting NETs and Treg interactions could offer a potential strategy for preventing HCC in patients with NASH.

Lay summary: Regulatory T-cells (Tregs) can promote tumor development by suppressing cancer immunosurveillance, but their role in carcinogenesis during non-alcoholic steatohepatitis (NASH) progression is unknown. Herein, we discovered that selectively increased intrahepatic Tregs can promote an immunosuppressive environment in NASH livers. Neutrophil extracellular traps (NETs) link innate and adaptive immunity by promoting Treg differentiation via metabolic reprogramming of naïve CD4+ T-cells. This mechanism could be targeted to prevent liver cancer in patients with NASH.

Keywords: Carcinogenesis; Hepatocellular carcinoma (HCC); Metabolic reprogramming; Neutrophil extracellular traps (NETs); Nonalcoholic steatohepatitis (NASH); Oxidative phosphorylation (OXPHOS); Regulatory T-cell (Treg).

Copyright © 2021. Published by Elsevier B.V.

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Conflict of interest statement

Conflict of interest The authors declare that they have no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details.

Figures

Fig. 1.

Fig. 1.. Tregs selectively increase during the premalignant period in NASH livers.

(A) Composition of key immune cells in STAM livers (age = 12 weeks, n = 5). (B) The proportion of total CD4+ T-cells (n = 5). (C) The proportion of intrahepatic Tregs (n = 5). (D) Absolute cell counts of intrahepatic Th1 cells, Th17 cells, and Tregs (n = 5). (E) Immunochemical staining of FoxP3+ cells (age = 12 weeks, n = 5). (F) Change of intrahepatic Tregs over time (n = 3). (G, H) Hepatic Tregs in DEN-HFCD and WD mouse models (n = 4-5). (I) The peripheral (spleen and blood) Tregs in WD mice at 12 weeks (n = 5). (J) Suppression assay of intrahepatic Tregs (n = 3). (K) Immunochemical staining of FoxP3 in the adjacent liver tissue of patients with NASH-HCC (n = 19), healthy liver (n = 19), and patients with NASH (n = 8). (A-J) Data from 1 representative experiment of 3 independent experiments are shown. **p <0.01, ***p <0.001, ****p <0.0001. n.s., no significance. Mean ± SEM. Student’s t test (B, C, E, G, H, I), two-way ANOVA (D, F, J), one-way ANOVA (K). DEN-HFCD, choline-deficient, high-fat diet+diethylnitrosamine; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; STAM, stelic animal model; Treg, regulatory T-cell; WD, western diet.

Fig. 2.

Fig. 2.. Inhibition of Tregs prevents HCC initiation and development in NASH livers.

(A) Experimental design of DT injection in the premalignant period. (B,C) Representative liver pictures at 20 weeks old, following Treg depletion in STAM mice using DT (B) (n = 7) or anti-CD25 antibodies (C) (n = 5). (D) Representative liver pictures of surface tumor nodes at 16 weeks in DEN-HFCD mice (n = 7). (E) Intrahepatic CD4+IFN-γ+ Th1 cells in the premalignant process (age = 12 weeks, n = 6). (F, G) TNF-α or IFN-γ secreting CD8+ T-cells in tumor tissues (age = 20 weeks, n = 5-6). (H) Survival analysis of STAM with or without DT treatment (n = 6, p <0.05). (I) Survival analysis of STAM with or without anti-CD25 antibody treatment (n = 8-9, p <0.01). The survival analysis was determined using the Kaplan-Meier method and the log-rank test. (H, I) Data from 1 representative experiment of 3 independent experiments are shown. **p <0.01, ***p <0.001, ****p <0.0001. Mean ± SEM. Student’s t test (B, C, D, E, G), two-way ANOVA (F). DEN-HFCD, choline-deficient, high-fat diet+diethylnitrosamine; DT, diphtheria toxin; DTR, diphtheria toxin receptor; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; STAM, stelic animal model; Th1, T helper type 1; Treg(s), regulatory T-cell(s).

Fig. 3.

Fig. 3.. A positive correlation between NETs and Tregs in NASH-HCC liver and NETs promote iTreg differentiation in vitro.

(A) The co-localization of NETs and Tregs (red, FoxP3; orange, Ly6G; NETs structure: green and blue double-staining). (B) Correlation between serum MPO-DNA and intrahepatic Tregs in STAM (age = 10-11 weeks, n = 20, Spearman’s coefficient r = 0.5686, p = 0.0089). (C) Immunohistochemical staining of the livers from patients with NASH-HCC (yellow arrows: NETs structure; red arrows: FoxP3+ cells). Patient A represents patients with relatively high Cit-H3 expression, while Patient B represents patients with low Cit-H3 expression. (D) Correlation analysis of Cit-H3 expression and numbers of Tregs in the livers of patients with NASH or NASH-HCC (n = 27, Spearman’s coefficient r = 0.6358, p = 0.0004). (E) In vitro, iTreg differentiation with NET or vehicle treatment (n = 3). (F) Suppressive assay of Tregs pre-treated with pure NETs (n = 3). (E, F) Data from 1 representative experiment of 3 independent experiments are shown. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001. Mean ± SEM. Two-way ANOVA (E, F). Repeated measurements were analyzed by a linear mixed-effect model for the comparison between different doses of TGF-β in E (p = 0.028). HCC, hepatocellular carcinoma; iTreg(s), induced Treg(s); NASH, non-alcoholic steatohepatitis; NET(s), neutrophil extracellular trap(s); STAM, stelic animal model; Treg(s), regulatory T-cell(s).

Fig. 4.

Fig. 4.. Transcriptome analysis of NET-treated naïve CD4+ T-cells.

(A) Heat map of DEGs (_p_adj <0.05) in naïve CD4+ T-cells cultured with or without NETs for 72 h (n = 3). (B) Heat map of T-cell activity-related genes in DEGs. (C) Volcano plot of DEGs in mice with all T-cell activity-related genes circled out. (D) KEGG enrichment analysis of most DEGs (absolute _log2 fold change_ >0.2). (E) GO analysis of most DEGs (absolute log 2 fold change >0.2). DEGs, differentially expressed genes; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NETs, neutrophil extracellular traps; Teff(s), effector T-cell(s); Treg(s), regulatory T-cell(s).

Fig. 5.

Fig. 5.. NETs promote Treg differentiation by enhancing mitochondrial OXPHOS in naïve CD4+ T-cells.

(A) Diagram and heat map of DEGs in the OXPHOS pathway. (B) Western blotting validation of mitochondrial complex I–V expression in naïve CD4+ T-cells (n = 4). (C) Mitochondrial complex I activity of naïve CD4+ T-cells (n = 4). (D) OCR of naïve CD4+ T-cells assessed by Seahorse (n = 5). (E) ECAR rate of naïve CD4+ T-cells (n = 5). (F) iTreg differentiation in NET-treated or control group with or without rotenone treatment (n = 4). (C, F) Data from 1 representative experiment of 3 independent experiments are shown. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001. n.s., no significance. Mean ± SEM. Student’s t test (C, E), two-way ANOVA (B, D, F). DEGs, differentially expressed genes; ECAR, extracellular acidification rate; iTreg(s), induced Treg(s); OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation; NET(s), neutrophil extracellular trap(s); Treg(s), regulatory T-cell(s).

Fig. 6.

Fig. 6.. NETs reprogram the metabolic process of naïve CD4+ T-cells through TLR4.

(A) Top receptor and cytokine pathway enrichment analysis from the Reactome database based on DEGs. (B) Immunofluorescence staining for TLR4 on naïve CD4+ T-cells (neutrophils: positive control). (C) OCR measured by Seahorse (n = 5). (D) Induced regulatory T-cell differentiation assessed in _Tlr4_WT and _Tlr4_−/− naïve CD4+ T-cells with NETs or vehicle treatment (n = 5). (D) Data from 1 representative experiment of 3 independent experiments are shown. ****p <0.0001. Mean ± SEM. Two-way ANOVA (C, D). NET(s), neutrophil extracellular trap(s); OCR, oxygen consumption rate; WT, wild-type.

Fig. 7.

Fig. 7.. Blocking NET formation reduces Treg activity and tumor formation in NASH livers.

(A) Western blotting analysis of Cit-H3 in STAM (n = 5). (B) Intrahepatic Tregs in STAM with or without DNase treatment (n = 4). (C) Proportion of CD4+ T-cells that are Tregs in STAM _Pad4_WT and STAM _Pad4_−/− mice (n = 5). (D,E) The hepatic Treg in WD mice (D) (n = 4) or DEN-HFCD mice (E) (n = 5) after NET depletion. (F) Suppressive capacity of intrahepatic Tregs (n = 3). (G, H) Liver tumor initiation in STAM or DEN-HFCD mice after NET depletion (n = 7). (A-F) Data from 1 representative experiment of 3 independent experiments are shown. **p <0.01, ***p <0.001, ****p <0.0001. Mean ± SEM. Student’s t test (A-E, H), two-way ANOVA (F), one-way ANOVA (G). DEN-HFCD, choline-deficient, high-fat diet+diethylnitrosamine; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; NET(s), neutrophil extracellular trap(s); STAM, stelic animal model; Treg(s), regulatory T-cell(s); WD, western diet.

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