CCL8-mediated recruitment of cytotoxic lymphocytes by P16-positive cells promotes adverse post-ischemic cardiac remodeling

BACKGROUND: Ischemic heart disease remains a leading cause of mortality worldwide, with adverse remodeling post-myocardial infarction (MI) driven by inflammation and cardiomyocyte loss. While cytotoxic lymphocytes exacerbate injury, and p16 marks cellular senescence in diseased hearts, the cell-type-specific roles of P16+ populations remain undefined. METHODS: Using p16-CreER;R26-tdT reporter mice, we mapped P16+ cellular heterogeneity post-MI. Senolytic effects were assessed via combination of dasatinib and quercetin (DQ) administration. Transcriptomic profiling (bulk/scRNA-seq) of sorted P16+ cells identified secreted factors, validated by in silico predictions and qPCR. Intercellular communication was analyzed using CellChat. Functional roles were tested via CCL8 neutralization, genetic Ccl8 ablation in P16+ cells, lymphocyte depletion, and intersectional genetic ablation of P16+ fibroblasts/macrophages using dual-recombinase systems (p16-DreER;Pdgfra-CreER;R26-lr-tdT-DTR and p16-DreER;Cx3cr1-CreER;R26-lr-tdT-DTR). RESULTS: P16 was induced in fibroblasts, macrophages, coronary endothelial cells, and cardiomyocytes post-MI. DQ treatment selectively eliminated P16+ macrophages and fibroblasts, improving cardiac function. Transcriptomics revealed P16+ macrophages and fibroblasts as primary CCL8 sources. CCL8 blockade reduced infiltration of cytotoxic lymphocytes (CD8+ T cells and NK cells), decreased cardiomyocyte apoptosis, and improved repair. Genetic Ccl8 ablation in P16+ cells replicated these benefits. Crucially, intersectional genetic ablation of P16+ fibroblasts, but not macrophages, reduced fibrosis and enhanced function, whereas CD8+ T cell (not NK cell) depletion attenuated remodeling. CONCLUSIONS: P16+ cells orchestrate adverse post-MI remodeling via CCL8-dependent recruitment of cytotoxic lymphocytes, expecially CD8+ T cells, driving cardiomyocyte apoptosis. Precision targeting of P16+ fibroblasts or CCL8 blockade represents a promising therapeutic strategy for ischemic heart disease. Overall design: p16-2A-CreER;R26-mTmG mice underwent myocardial infarction (MI) surgery, received tamoxifen via oral gavage 5 days post-MI. Non-cardiomyocyte cells were harvested from the cardiac infarct zone on day 7 to prepare a single-cell suspension. GFPhiPDGFRahi,GFPloPDGFRahi and GFPhiF4/80hi,GFPloF4/80hi cells were subsequently isolated by flow cytometry sorting (FACS) and analyzed using RNA sequencing (RNA-seq).

研究背景：缺血性心脏病仍是全球范围内的主要致死病因，心肌梗死（myocardial infarction，MI）后的不良重构由炎症反应与心肌细胞丢失所驱动。尽管细胞毒性淋巴细胞会加重心肌损伤，且p16可作为病变心脏中细胞衰老的标记物，但P16阳性（P16+）细胞群的细胞类型特异性功能仍未明确。
实验方法：本研究使用p16-CreER;R26-tdT报告小鼠，绘制了心肌梗死后P16阳性细胞的异质性图谱。通过达沙替尼（dasatinib）与槲皮素（quercetin）联合给药（DQ）评估了衰老裂解疗法的效果。对分选得到的P16阳性细胞进行转录组分析（批量/单细胞RNA测序），鉴定其分泌的细胞因子，并通过计算机预测与实时定量PCR（qPCR）进行验证。使用CellChat工具分析细胞间通讯网络。通过CCL8中和、P16阳性细胞中Ccl8基因敲除、淋巴细胞清除，以及使用双重组酶系统（p16-DreER;Pdgfra-CreER;R26-lr-tdT-DTR与p16-DreER;Cx3cr1-CreER;R26-lr-tdT-DTR）对P16阳性成纤维细胞/巨噬细胞进行交叉遗传敲除，验证相关细胞的功能作用。
实验结果：研究发现，心肌梗死后成纤维细胞、巨噬细胞、冠状动脉内皮细胞与心肌细胞中均出现P16表达上调。DQ治疗可选择性清除P16阳性巨噬细胞与成纤维细胞，改善心脏功能。转录组分析显示，P16阳性巨噬细胞与成纤维细胞是CCL8的主要分泌来源。阻断CCL8可减少细胞毒性淋巴细胞（CD8+ T细胞与自然杀伤（NK）细胞）的浸润，降低心肌细胞凋亡，促进损伤修复。在P16阳性细胞中敲除Ccl8基因可重现上述保护效应。关键的是，交叉遗传敲除P16阳性成纤维细胞（而非巨噬细胞）可减轻心肌纤维化并改善心脏功能，而清除CD8+ T细胞（而非NK细胞）可缓解不良重构。
研究结论：P16阳性细胞通过依赖CCL8的方式招募细胞毒性淋巴细胞（尤其是CD8+ T细胞），介导心肌细胞凋亡，从而调控心肌梗死后的不良重构。精准靶向P16阳性成纤维细胞或阻断CCL8，有望成为缺血性心脏病的新型治疗策略。
实验整体设计：将p16-2A-CreER;R26-mTmG小鼠进行心肌梗死（MI）造模，于造模后第5天通过灌胃给予他莫昔芬。于造模后第7天从心脏梗死区分离非心肌细胞，制备单细胞悬液。随后通过流式细胞分选（flow cytometry sorting，FACS）分离GFP高表达/血小板衍生生长因子受体α（PDGFRα）高表达（GFPhiPDGFRahi）、GFP低表达/PDGFRα高表达（GFPloPDGFRahi）以及GFP高表达/F4/80高表达（GFPhiF4/80hi）、GFP低表达/F4/80高表达（GFPloF4/80hi）的细胞，并进行RNA测序（RNA-seq）分析。