Medical Microbiology, Interdisciplinary Program, Graduate School, Chulalongkorn University, Bangkok, Thailand.
Mycology Unit, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
PLoS One. 2023 Jan 24;18(1):e0280565. doi: 10.1371/journal.pone.0280565. eCollection 2023.
Neutrophils are innate immune cells that play crucial roles in response to extracellular pathogens, including bacteria and fungi. Pythium insidiosum (P insidiosum) is a fungus-like pathogen that causes "pythiosis" in mammals. This study investigated in vitro function of human neutrophils against P. insidiosum. We demonstrated the killing mechanism of neutrophils when incubated with P. insidiosum zoospores (infective stage), such as phagocytosis and neutrophil extracellular traps (NETs). Healthy human neutrophils significantly reduced six strains of live zoospores isolated from different sources compared to the condition without neutrophils (p < 0.001), observed by colony count and trypan blue staining. As our results showed the killing ability of neutrophils, we further investigated the neutrophil killing mechanism when incubating with zoospores. Our study found that only two strains of heat-killed zoospores significantly induced phagocytosis (p < 0.01). Co-culture of heat-killed zoospores and neutrophils demonstrated NET formation, which was detected by immunofluorescence staining using DAPI, anti-myeloperoxidase, and anti-neutrophil elastase and quantitated under the fluorescence microscope. In addition, the level of cell-free DNA released from neutrophils (as a marker of NET production) after incubation with zoospores showed significantly increased levels when compared with unstimulated neutrophils (p < 0.001). Our findings demonstrate that neutrophils revealed the NET formation in response to P. insidiosum zoospores. This study is the first observation of the neutrophil mechanism against P. insidiosum, which could provide a better understanding of some parts of the innate immune response during pythiosis.
中性粒细胞是先天免疫系统的细胞，在应对包括细菌和真菌在内的细胞外病原体方面发挥着至关重要的作用。Pyhtium insidiosum（P insidiosum）是一种真菌样病原体，可在哺乳动物中引起“pyhtiosis”。本研究调查了人中性粒细胞对 P. insidiosum 的体外功能。我们证明了中性粒细胞与 P. insidiosum 游动孢子（感染阶段）孵育时的杀伤机制，如吞噬作用和中性粒细胞细胞外陷阱（NETs）。与没有中性粒细胞的情况相比，健康的人中性粒细胞显著减少了从不同来源分离的六种活游动孢子（p < 0.001），通过菌落计数和台盼蓝染色观察到。由于我们的结果表明了中性粒细胞的杀伤能力，因此我们进一步研究了与游动孢子孵育时中性粒细胞的杀伤机制。我们的研究发现，只有两种热灭活的游动孢子显著诱导了吞噬作用（p < 0.01）。热灭活的游动孢子和中性粒细胞共培养显示 NET 形成，通过使用 DAPI、抗髓过氧化物酶和抗中性粒细胞弹性蛋白酶的免疫荧光染色检测到，并在荧光显微镜下定量。此外，与未刺激的中性粒细胞相比，与游动孢子孵育后从中性粒细胞释放的无细胞 DNA 水平（作为 NET 产生的标志物）显著增加（p < 0.001）。我们的发现表明，中性粒细胞对 P. insidiosum 游动孢子显示出 NET 形成。本研究首次观察到中性粒细胞针对 P. insidiosum 的机制，这可以更好地了解 pyhtiosis 期间先天免疫反应的某些部分。
