工程菌调控肿瘤微环境是肿瘤治疗领域的一个前沿交叉方向，细菌经过改造后可以编码并局部递送多种有效物质。当工程菌进入肿瘤微环境时会受到免疫系统的识别，与巨噬细胞发生互作，因此了解细菌与巨噬细胞互作过程中的变化是我们开展工程菌研究的基础与前提。巨噬细胞作为先天性免疫防线的重要组成部分，已有广泛的研究报道其在应对外源感染时发挥的免疫功能，但对于免疫反应相关信号通路和物质代谢的时序变化尚缺乏系统理解。

本文研究了鼠伤寒沙门氏菌SL1344感染巨噬细胞RAW 264.7后在基因和代谢水平上发生的时序变化，旨在全面了解感染期间的动态分子网络。在鼠伤寒沙门氏菌感染巨噬细胞的0 h、8 h和16 h收集细胞样本进行转录物组测序（RNA-sequencing，RNA-seq）与飞行时间二次离子质谱（TOF-SIMS）检测，通过对转录物组数据进行分析，包括差异基因表达、聚类、通路注释、分子网络研究，对代谢组数据进行离子差异分析、代谢物标注、代谢网络研究，来阐明巨噬细胞信号通路与代谢物质的变化。研究发现感染的巨噬细胞在形态、转录和代谢水平上都发生了明显变化。差异基因分析确定了显著的上调和下调模式，通过聚类揭示了六个基因簇，涉及多种信号通路，包括免疫反应、膜转运和脂质代谢等。代谢物同样存在上下调表达模式，其中可注释的代谢物大部分集中在糖、脂类、蛋白质和核酸等代谢物中。

本文研究发现巨噬细胞对沙门氏菌感染表现出动态响应，具有明显的基因和代谢时序表达模式。免疫反应、膜转运和脂质代谢通路的协同激活暗示了巨噬细胞对外源感染的多层次细胞适应性，脂质、糖类、蛋白质、核酸及相关小分子在能量代谢与物质循环中的变化进一步证实了部分巨噬细胞信号通路变化对免疫反应的支撑作用。本研究建立了使用转录物组技术、空间代谢组技术观测沙门氏菌感染巨噬细胞的组学方法，为巨噬细胞响应沙门氏菌感染的分子机制提供了重要见解，对进一步设计工程菌调控肿瘤微环境具有重要意义。

The regulation of the tumor microenvironment by engineered bacteria represents a cutting-edge interdisciplinary approach in tumor therapy, where modified bacteria can encode and locally deliver a variety of effective substances. Upon entering the tumor microenvironment, engineered bacteria are recognized by the immune system and interact with macrophages. Therefore, understanding the changes occurring during the interaction between bacteria and macrophages is fundamental and a prerequisite for conducting research on engineered bacteria. Macrophages, as an integral part of the innate immune defense, have been extensively studied for their immune functions in response to exogenous infections. However, a systematic understanding of the temporal changes in immune response-related signaling pathways and substance metabolism is lacking.

This study investigated the temporal changes occurring at the gene and metabolic levels in RAW 264.7 macrophages infected with Salmonella Typhimurium SL1344, aiming to comprehensively understand the dynamic molecular network during infection. Cell samples were collected at 0 h, 8 h, and 16 h post-infection with Salmonella Typhimurium, and subjected to RNA sequencing（RNA-seq）and time-of-flight secondary ion mass spectrometry（TOF-SIMS）analysis. Transcriptome data were analyzed upstream and downstream, including differential gene expression, clustering, pathway annotation, and molecular network analysis. Metabolome data were analyzed for ion differential expression, metabolite annotation, and metabolic network analysis to elucidate changes in macrophage signaling pathways and metabolites. The study revealed significant morphological, transcriptional, and metabolic changes in infected macrophages. Differential gene analysis identified significant upregulation and downregulation patterns, and clustering revealed six gene clusters involving multiple signaling pathways, including immune response, membrane transport, and lipid metabolism, among others. Similarly, metabolites exhibited upregulation and downregulation patterns, with most annotatable metabolites concentrated in sugars, lipids, proteins, and nucleic acids.

Macrophages respond dynamically to Salmonella infection, exhibiting distinct gene and metabolic temporal expression patterns. The coordinated activation of immune response, membrane transport, and lipid metabolism pathways suggests multilayered cellular adaptability of macrophages to exogenous infections. Changes in lipids, carbohydrates, proteins, nucleic acids, and related small molecules in energy metabolism and substance circulation further confirm the supportive role of certain macrophage signaling pathway changes in immune response. This study establishes an omics approach using transcriptomics and spatial metabolomics to observe Salmonella-infected macrophages, providing significant insights into the molecular mechanisms of macrophage responses to Salmonella infection and offering valuable implications for further design of engineered bacteria to regulate the tumor microenvironment.