Bacterial infections shape cardiac macrophages' response to ischemia

Background: Patients with bacterial infections are at increased risk for subsequent cardiovascular events. Whether infections' effects on innate immune cells within the cardiovascular system influence subsequent pathologies remains unclear. Here, we explore cardiac myeloid cells' chronic adaptations to a preceding bacterial insult and implications for subsequent myocardial ischemia. Methods: We used various flow cytometry protocols to assess cardiac immune cells, peripheral leukocytes and hematopoietic stem and progenitor cells in bone marrow and spleen. A genetic fate mapping model was used to determine cardiac macrophages' origin after bacteremia. Cardiac leukocytes were analyzed using scRNAseq. Nanoparticle-mediated RNAi was used to target macrophages in-vivo. Results: Cardiac macrophage numbers increased sharply, and numerical changes alongside subset alterations persisted over time. Fate mapping pointed towards local origin as the primary macrophage source after infection. Profiling macrophage heterogeneity using scRNAseq, we identified two previously unknown subpopulations remaining after resolution of infection. Whereas heightened metabolic activity was one subset's primary feature, the other displayed excessive chemotactic properties, which amplified cardiac leukocyte recruitment and inflammation after a subsequent ischemic injury. Targeting cardiac macrophages' surplus inflammatory activity after infection using nanoparticle-enabled, macrophage-directed RNAi kept disproportionate subsequent ischemic inflammation at bay. Conclusions: Bacteremia induces long-lasting changes in the cardiovascular system's innate immune cells' composition. This may amplify myocardial inflammation after a subsequent ischemic injury. Overall design: C57Bl6/J mice were subject to cecal ligation and puncture (CLP); 6 weeks later, animals were sacrificed and hearts were flushed and digested. From Single cell dispension, CD45 positive cells were isolated by fluorescence activated cell sorting and immediately prepped for single cell sequencing by 10x genomics workflow. Animals of the same age without treatment were used as a control.

研究背景：细菌感染患者发生后续心血管不良事件的风险显著升高。目前尚不清楚感染对心血管系统内固有免疫细胞的作用是否会影响后续病理进程。本研究旨在探讨心脏髓系细胞对先前细菌感染损伤的慢性适应性变化，以及其对后续心肌缺血的影响。
研究方法：本研究采用多种流式细胞术方案，评估心脏免疫细胞、外周白细胞以及骨髓与脾脏中的造血干细胞及祖细胞。使用遗传命运图谱模型明确菌血症后心脏巨噬细胞的来源。通过单细胞RNA测序（single-cell RNA sequencing，scRNAseq）分析心脏白细胞。采用纳米颗粒介导的RNA干扰（RNA interference，RNAi）在体靶向巨噬细胞。
研究结果：心脏巨噬细胞数量急剧升高，且数量变化伴随亚群改变可长期持续。命运图谱分析显示，感染后心脏巨噬细胞的主要来源为局部增殖。通过scRNAseq分析巨噬细胞异质性，我们鉴定出感染消退后残留的两个此前未被发现的巨噬细胞亚群。其中一个亚群以代谢活性增强为主要特征，另一个则表现出过度的趋化特性，该特性可在后续缺血性损伤时加剧心脏白细胞招募与炎症反应。采用纳米颗粒介导的巨噬细胞靶向RNAi干预感染后心脏巨噬细胞的过度炎症活性，可抑制后续不成比例的缺血性炎症反应。
研究结论：菌血症可诱导心血管系统固有免疫细胞组成发生长期改变，该改变可能在后续缺血性损伤时加剧心肌炎症反应。
实验整体设计：对C57Bl6/J小鼠实施盲肠结扎穿刺术（cecal ligation and puncture，CLP）；6周后处死小鼠，对心脏进行冲洗与组织消化。制备单细胞悬液后，通过荧光激活细胞分选分离CD45阳性细胞，并立即采用10x Genomics测序流程完成单细胞测序文库制备。选取同年龄未接受任何处理的小鼠作为对照。