Division of Inflammation and Infection, Department of Biomedical and Clinical Sciences, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden.
Front Immunol. 2024 Nov 6;15:1454881. doi: 10.3389/fimmu.2024.1454881. eCollection 2024.
Helminth coinfection with tuberculosis (TB) can alter the phenotype and function of macrophages, which are the major host cells responsible for controlling (Mtb). However, it is not known whether helminth infection stimulates the release of host-derived extracellular vesicles (EVs) to induce or maintain their regulatory network that suppresses TB immunity. We previously showed that pre-exposure of human monocyte-derived macrophages (hMDMs) with protein antigens (ASC) results in reduced Mtb infection-driven proinflammation and gained bacterial control. This effect was entirely dependent on the presence of soluble components in the conditioned medium from helminth antigen-pre-exposed macrophages.
Our objective was to investigate the role of EVs released from helminth antigen-exposed hMDMs on Mtb-induced proinflammation and its effect on Mtb growth in hMDMs. Conditioned medium from 48-h pre-exposure with ASC or antigen (SM) was used to isolate EVs by ultracentrifugation. EVs were characterized by immunoblotting, flow cytometry, nanoparticle tracking assay, transmission electron microscopy, and a total of 377 microRNA (miRNA) from EVs screened by TaqMan array. Luciferase-expressing Mtb H37Rv was used to evaluate the impact of isolated EVs on Mtb growth control in hMDMs.
EV characterization confirmed double-membraned EVs, with a mean size of 140 nm, expressing the classical exosome markers CD63, CD81, CD9, and flotillin-1. Specifically, EVs from the ASC conditioned medium increased the bacterial control in treatment-naïve hMDMs and attenuated Mtb-induced IL-1β at 5 days post-infection. Four miRNAs showed unique upregulation in response to ASC exposure in five donors. Pathway enrichment analysis showed that the MAPK and PI3K-AKT signaling pathways were regulated. Among the mRNA targets, relevant for regulating inflammatory responses and cellular stress pathways, CREB1 and MAPK13 were identified. In contrast, SM exposure showed significant regulation of the TGF-β signaling pathway with SMAD4 as a common target.
Overall, our findings suggest that miRNAs in EVs released from helminth-exposed macrophages regulate important signaling pathways that influence macrophage control of Mtb and reduce inflammation. Understanding these interactions between helminth-induced EVs, miRNAs, and macrophage responses may inform novel therapeutic strategies for TB management.
蠕虫感染与结核病（TB）的合并感染会改变巨噬细胞的表型和功能，巨噬细胞是控制（Mtb）的主要宿主细胞。然而，目前尚不清楚蠕虫感染是否会刺激宿主来源的细胞外囊泡（EVs）的释放，以诱导或维持抑制 TB 免疫的调节网络。我们之前的研究表明，人类单核细胞来源的巨噬细胞（hMDM）在暴露于 蛋白抗原（ASC）之前，可减少 Mtb 感染驱动的促炎反应和获得细菌控制。这种效应完全依赖于从蠕虫抗原预处理巨噬细胞的条件培养基中存在可溶性成分。
我们的目标是研究从蠕虫抗原暴露的 hMDM 释放的 EVs 在 Mtb 诱导的促炎反应及其对 hMDM 中 Mtb 生长的影响。使用 48 小时预孵育 ASC 或 抗原（SM）的条件培养基通过超速离心分离 EVs。通过免疫印迹、流式细胞术、纳米颗粒跟踪分析、透射电子显微镜和 TaqMan 阵列筛选的总共 377 种 microRNA（miRNA）对 EVs 进行了表征。使用表达荧光素酶的 Mtb H37Rv 来评估分离的 EVs 对 hMDM 中 Mtb 生长控制的影响。
EVs 的特征确认了双层膜 EVs，平均大小为 140nm，表达经典的外泌体标记物 CD63、CD81、CD9 和 flotillin-1。具体而言，ASC 条件培养基中的 EVs 增加了治疗前 hMDM 中的细菌控制，并在感染后 5 天减弱了 Mtb 诱导的 IL-1β。在五个供体中，有四种 miRNA 对 ASC 暴露表现出独特的上调。途径富集分析表明，MAPK 和 PI3K-AKT 信号通路受到调节。在与调节炎症反应和细胞应激途径相关的 mRNA 靶标中，鉴定出 CREB1 和 MAPK13。相比之下，SM 暴露显示 TGF-β 信号通路的显著调节，SMAD4 是共同的靶标。
总体而言，我们的研究结果表明，从蠕虫暴露的巨噬细胞中释放的 EVs 中的 miRNA 调节影响 Mtb 控制和减少炎症的重要信号通路。了解这些蠕虫诱导的 EVs、miRNA 和巨噬细胞反应之间的相互作用可能为 TB 管理提供新的治疗策略。
