Takii Takemasa, Yamada Hiroyuki, Motozono Chihiro, Yamasaki Sho, Torrelles Jordi B, Turner Joanne, Kimishima Aoi, Asami Yukihiro, Ohara Naoya, Hida Shigeaki, Hayashi Hidetoshi, Onozaki Kikuo
Department of Mycobacterium Reference and Research, the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo, Japan.
Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan.
mSphere. 2025 Jun 25;10(6):e0011025. doi: 10.1128/msphere.00110-25. Epub 2025 May 19.
We previously reported that live, but not dead, virulent () H37Rv bacilli induce cell death in human lung fibroblast cell lines, MRC-5, MRC-9, and TIG-1. Here, using two distinct strains from two different lineages (HN878 lineage 2 and H37Rv lineage 4), we confirmed cell death at day 2 after infection with a device that measures cell growth/cytotoxicity in real time (Maestro-Z [AXION]). bacilli uptake by the fibroblast was confirmed with a transmission electron microscope on day 2. Expressions of inflammatory cytokines and interleukin (IL)-1β, IL-6, and IL-8 were observed when exposed to live, but not dead bacteria. The cell death of fibroblasts induced by both strains tested was prevented by caspase-1/4 and NLRP3 inflammasome inhibitors, but not by caspase-3 and caspase-9 inhibitors. Therefore, we classified the fibroblast cell death by infection as pyroptosis. To investigate the biological and pathological relevance of fibroblast cell death by infection, we performed dual RNA-Seq analysis on within fibroblasts and -infected fibroblasts at day 2. In bacilli , , , and genes were highly induced during infection. These genes play roles in survival in a hypoxic environment, production of a calcium-binding protein-inducing cytokine, and regulation of transcription in a toxin-antitoxin system. The gene expressions of IL-1β, IL-6, and IL-8, caspase-4, and NLRP3, but not of caspase-3 and caspase-9, were augmented in bacilli-infected fibroblasts. Taken together, our study suggests that bacilli attempt to survive in lung fibroblasts and that pyroptosis of the host fibroblasts activates the immune system against the infection.
The role of "non-classical immune cells," that is, fibroblasts, epithelial cells, adipocytes, etc., except for the "classical immune cells," that is, macrophages and lymphoid cells, is not well known in the infection of Mtb bacilli. We have previously found that live, but not dead, Mtb bacilli induce cell death in human lung fibroblasts, except in human macrophages and monocytes. The present study reveals that fibroblasts ingest Mtb bacilli the same as macrophages and that Mtb bacilli within fibroblasts attempt to survive in the host cells, and pyroptosis, including the production of inflammatory cytokines, is induced in the Mtb-infected fibroblasts. Our results suggest that pyroptosis of the host fibroblasts activates the immune system against the infection.
我们之前报道过，活的而非死的强毒力结核分枝杆菌H37Rv可诱导人肺成纤维细胞系MRC - 5、MRC - 9和TIG - 1发生细胞死亡。在此，我们使用来自两个不同谱系的两种不同结核分枝杆菌菌株（HN878谱系2和H37Rv谱系4），通过一种可实时测量细胞生长/细胞毒性的设备（Maestro - Z [AXION]）证实了感染后第2天细胞死亡。在第2天用透射电子显微镜证实了成纤维细胞对结核分枝杆菌的摄取。当暴露于活细菌而非死细菌时，观察到炎性细胞因子以及白细胞介素（IL）-1β、IL - 6和IL - 8的表达。测试的两种结核分枝杆菌菌株诱导的成纤维细胞死亡可被半胱天冬酶 - 1/4和NLRP3炎性小体抑制剂阻止，但不能被半胱天冬酶 - 3和半胱天冬酶 - 9抑制剂阻止。因此，我们将结核分枝杆菌感染诱导的成纤维细胞死亡归类为细胞焦亡。为了研究结核分枝杆菌感染诱导的成纤维细胞死亡的生物学和病理学相关性，我们在第2天对未感染和感染结核分枝杆菌的成纤维细胞内的结核分枝杆菌进行了双RNA测序分析。在结核分枝杆菌感染期间，基因、、和被高度诱导。这些基因在低氧环境下的存活、钙结合蛋白诱导细胞因子的产生以及毒素 - 抗毒素系统中的转录调控中发挥作用。在感染结核分枝杆菌的成纤维细胞中，IL - 1β、IL - 6和IL - 8、半胱天冬酶 - 4和NLRP3的基因表达增加，但半胱天冬酶 - 3和半胱天冬酶 - 9的基因表达未增加。综上所述，我们的研究表明结核分枝杆菌试图在肺成纤维细胞中存活，并且宿主成纤维细胞的细胞焦亡激活了针对感染的免疫系统。
“非经典免疫细胞”，即除“经典免疫细胞”（巨噬细胞和淋巴细胞）之外的成纤维细胞、上皮细胞、脂肪细胞等，在结核分枝杆菌感染中的作用尚不明确。我们之前发现，活的而非死的结核分枝杆菌可诱导人肺成纤维细胞发生细胞死亡，人类巨噬细胞和单核细胞除外。本研究揭示成纤维细胞与巨噬细胞一样摄取结核分枝杆菌，并且成纤维细胞内的结核分枝杆菌试图在宿主细胞中存活，并且在感染结核分枝杆菌的成纤维细胞中诱导了包括炎性细胞因子产生在内的细胞焦亡。我们的结果表明宿主成纤维细胞的细胞焦亡激活了针对感染的免疫系统。
