Imai Takashi, Miyazaki Shinya, Miyazaki Yukiko, Kagaya Wataru, Nakashima Mai, Ito Kumpei, Sase Miho, Yoshioka-Takeda Misato, Shimokawa Chikako, Hayashi Kyoko, Araki Tamasa, Annoura Takeshi, Itokawa Kentaro, Komagata Osamu, Ha Ngo-Thanh, Shimo Aoi, Murakami Takashi, Hisaeda Hajime
Department of Parasitology, National Institute of Infectious Diseases (NIID), Japan Institute for Health Security (JIHS), Tokyo, Japan; Department of Infectious Diseases and Host Defense, Gunma University, Gunma, Japan; Department of Microbiology, Saitama Medical University, Saitama, Japan; Leprosy Research Center, NIID, JIHS, Tokyo, Japan; Antimicrobial Resistance Research Center, NIID, JIHS, Tokyo, Japan.
Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
Acta Trop. 2025 Sep 17;271:107843. doi: 10.1016/j.actatropica.2025.107843.
Although malaria parasites are traditionally known to infect enucleated red blood cells, recent evidence-including our previous work-demonstrates their ability to invade nucleated erythroid precursors (erythroblasts). However, visualizing and quantifying parasitized erythroblasts (pEBs) in vivo remains challenging due to their rarity in peripheral blood and the limitations of existing techniques. In this study, we employed imaging flow cytometry (ImageStream) to identify and characterize pEBs in mice infected with Plasmodium yoelii 17XNL expressing green fluorescent protein and compared with conventional flow cytometry (FACS). At 14 days post-infection, splenic, bone marrow and peripheral blood cells were stained with antibodies against TER119 (erythroid marker) and MHC class I, which is expressed on nucleated host cells. To confirm the presence of nuclei, we also used Hoechst 33342 DNA staining. ImageStream analysis enabled simultaneous detection of GFP⁺TER119⁺MHC class I Hoechst pEBs and morphological visualization. While no pEBs were detected in peripheral blood, a significant population was identified in the spleen and bone marrow. Importantly, quantification of pEB frequencies by ImageStream closely matched those obtained by FACS, validating the robustness of the imaging-based approach. Notably, ImageStream also revealed doublets and triplets involving pEBs and MHC class I⁺ cells, suggestive of erythroblastic islands or immunological interactions. Blebbing structures were occasionally observed, indicating apoptosis-like processes. These findings demonstrate that ImageStream offers a powerful platform for high-throughput, image-based detection of parasitized-erythroblasts in malaria. This approach may open new avenues for studying host-parasite interactions within hematopoietic tissues and their roles in parasite persistence and immune evasion.
尽管传统上认为疟原虫会感染无细胞核的红细胞，但最近的证据——包括我们之前的研究——表明它们有能力侵入有细胞核的红细胞前体（成红细胞）。然而，由于成红细胞在外周血中数量稀少以及现有技术的局限性，在体内可视化和定量被寄生的成红细胞（pEBs）仍然具有挑战性。在本研究中，我们采用成像流式细胞术（ImageStream）来识别和表征感染了表达绿色荧光蛋白的约氏疟原虫17XNL的小鼠体内的pEBs，并与传统流式细胞术（FACS）进行比较。感染后14天，用抗TER119（红细胞标志物）和MHC I类分子（在有细胞核的宿主细胞上表达）的抗体对脾脏、骨髓和外周血细胞进行染色。为了确认细胞核的存在，我们还使用了Hoechst 33342 DNA染色。ImageStream分析能够同时检测GFP⁺TER119⁺MHC I类分子⁺Hoechst⁺的pEBs并进行形态学可视化。虽然在外周血中未检测到pEBs，但在脾脏和骨髓中发现了大量pEBs。重要的是，通过ImageStream对pEB频率的定量与通过FACS获得的结果密切匹配，验证了基于成像方法的稳健性。值得注意的是，ImageStream还揭示了涉及pEBs和MHC I类分子⁺细胞的双联体和三联体，提示成红细胞岛或免疫相互作用。偶尔观察到泡状结构，表明存在类似凋亡的过程。这些发现表明，ImageStream为疟疾中被寄生的成红细胞的高通量、基于图像的检测提供了一个强大的平台。这种方法可能为研究造血组织内的宿主 - 寄生虫相互作用及其在寄生虫持续存在和免疫逃避中的作用开辟新途径。
