Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Medical Nanotechnology and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
BMC Microbiol. 2024 Apr 4;24(1):117. doi: 10.1186/s12866-024-03270-z.
Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission.
MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR.
All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups.
Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.
利什曼病作为一种被忽视的热带病（NTD），是由白蛉属沙蝇叮咬而感染利什曼原虫寄生虫引起的。在受感染的叮咬后，会发生一系列先天和适应性免疫反应，其中可以提到中性粒细胞是启动者。在这些战斗细胞的多种功能中，已经研究了在单独存在的大镰形前鞭毛体和白蛉属唾液腺匀浆（SGH）以及模拟自然传播条件的情况下，中性粒细胞细胞外陷阱（NETs）的形成。
在三个不同的组中研究了 L. major 和 SGH 对 NETs 形成的影响：中性粒细胞+SGH（NS）、中性粒细胞+L. major（NL）和中性粒细胞+L. major+SGH（NLS），同时设有阴性和阳性对照，在孵育后 2、4 和 6 小时进行检测。使用不同的显微镜方法来可视化包含 NETs 的结构：吖啶橙/溴化乙锭染色的荧光显微镜、吉姆萨染色的光学显微镜和扫描电子显微镜。此外，还通过实时 PCR 评估了三种不同基因 NE、MPO 和 MMP9 的表达水平。
所有三种显微镜方法都揭示了相似的结果，在 NS 组中，作为 NETosis 迹象的染色质外溢，在每个三个时间点都不是很明显；但是，在 NL 组，尤其是在 NLS 组中，观察到更多的 NETosis，并且中性粒细胞与前鞭毛体之间的相互作用在 NL 组中，以及在 NLS 组中与唾液的相互作用，随着时间的推移逐渐增加。实时显示，暴露后 2 和 4 小时，MPO、NE 和 MMP9 基因的表达增加，然后在大多数组中在 6 小时时减少。
因此，确定了在 NLS 组中同时存在寄生虫和唾液对 NETs 的形成产生比 NL 和 NS 组更大的影响。
