程序性死亡受体-1信号在压力负荷性心力衰竭中的作用及机制

PD-1 signaling modulates immune responses in pressure overload-induced heart failure via the TLR/NF-κB pathway

摘要

目的 探讨程序性死亡受体-1(programmed cell death protein 1, PD-1)信号在压力负荷性心力衰竭中的作用及其免疫调控机制。方法 分别在野生型(WT)和PD-1基因敲除小鼠中采用主动脉缩窄术(transverse aortic constriction, TAC)建立小鼠压力负荷性心力衰竭模型并进行比较。通过超声心动图评价心脏功能变化,HE及小麦胚凝集素(WGA)染色观察心肌形态学改变；流式细胞术分析心脏炎症细胞浸润特征；RNA测序检测心肌组织中炎症及免疫相关基因表达谱。体外实验中,采用坏死性心肌细胞提取物(necrotic cardiomyocyte extracts, NCEs)刺激小鼠腹腔来源炎症细胞(PDICs),并联合TLR2/TLR4阻断抗体或NF-κB抑制剂,探讨PD-1上调的分子机制。结果 RNA测序结果显示,TAC术后心肌中PD-1及其配体PD-L1表达显著上调(P＜0.000 1)。与WT组相比,PD-1敲除小鼠在TAC术后生存率明显下降(P=0.004 3),左心室射血分数(LVEF)(P=0.000 4)及短轴缩短率(FS)显著降低(P=0.000 6),心肌纤维化程度加重(P=0.017 7),心肌细胞横截面积增大(P=0.013 2),心重/体质量比(P＜0.000 1)、NT-proBNP(P=0.011 9)及肌钙蛋白I/T水平均升高(P=0.000 4)。流式细胞术结果显示,PD-1缺失显著增加心脏中CD64⁺巨噬细胞(P=0.007 8)及CD4⁺(P＜0.000 1)、CD8⁺T细胞浸润(P＜0.000 1)。体外实验表明,NCEs可通过激活TLR2/TLR4受体及下游NF-κB信号通路上调PDICs中PD-1表达,阻断TLR2/4或抑制NF-κB可显著减弱上述作用。结论 PD-1信号在压力负荷性心力衰竭中具有重要的免疫调节和心脏保护作用。PD-1通过抑制免疫细胞过度活化,减轻心脏炎症反应和心肌损伤,从而维持心脏功能稳定。其上调依赖于TLR2/TLR4-NF-κB通路的激活,而PD-1缺失可导致炎症反应失衡和心功能恶化。这为阐明心力衰竭的免疫机制及开发新的免疫调节治疗策略提供了实验依据。

Abstract

Objective To investigate the role and immunoregulatory mechanism of programmed cell death protein 1 (PD-1) signaling in pressure overload-induced heart failure. Methods A pressure overload heart failure model was established in wild-type (WT) and PD-1 knockout mice by transverse aortic constriction (TAC). Echocardiography was used to evaluate cardiac function. Histological changes were assessed by hematoxylin-eosin (HE) and wheat germ agglutinin (WGA) staining. Flow cytometry was performed to analyze cardiac immune cell infiltration, and RNA sequencing was used to examine the expression profile of inflammation- and immunity-related genes. In vitro, peritoneal-derived inflammatory cells (PDICs) from WT mice were stimulated with necrotic cardiomyocyte extracts (NCEs), with or without TLR2/TLR4 blocking antibodies or an NF-κB inhibitor, to explore the molecular mechanism of PD-1 upregulation. Results RNA sequencing revealed significant upregulation of PD-1 and PD-L1 in TAC-treated myocardium (P＜0.000 1). Compared with WT mice, PD-1 knockout mice showed decreased survival (P=0.004 3), reduced left ventricular ejection fraction (LVEF) and fractional shortening (FS) (P＜0.05), aggravated myocardial fibrosis (P=0.017 7), increased cross-sectional area of cardiomyocytes (P=0.013 2) and heart weight-body mass ratio (P＜0.000 1), and elevated serum NT-proBNP (P=0.011 9) and cardiac troponin I/T levels (P=0.000 4). Flow cytometry showed increased infiltration of CD64 ⁺ macrophages (P=0.007 8) and CD4 ⁺/CD8 ⁺ T cells in the PD-1 ^-/- group (P＜0.000 1). In vitro, NCEs upregulated PD-1 expression in PDICs via TLR2/TLR4-mediated activation of the NF-κB pathway, which was blocked by TLR2/4 antibodies or NF-κB inhibition. Conclusion PD-1 signaling plays a crucial role in modulating cardiac immune homeostasis and protecting against pressure overload-induced heart failure. By suppressing excessive immune activation and inflammation, PD-1 preserves cardiac structure and function. Its upregulation depends on the activation of the TLR2/TLR4-NF-κB pathway, while PD-1 deficiency exacerbates inflammation and cardiac dysfunction. These findings provide new insights into the immunopathogenesis of heart failure and suggest PD-1 as a potential therapeutic target.

关键词

程序性死亡受体-1(PD-1) / 心力衰竭 / 炎症反应 / 免疫调节 / TLR信号通路

Key words

programmed cell death protein 1 (PD-1) / heart failure / inflammatory response / immune regulation / TLR signaling pathway

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基金资助

国家自然科学基金青年科学基金项目(82400465)