Departamento de Sanidad Animal, Instituto de Ganadería de Montaña (IGM) CSIC-ULE, Grulleros, León, Spain.
Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24071, León, Spain.
Vet Res. 2023 Jul 18;54(1):61. doi: 10.1186/s13567-023-01193-7.
Neutrophils constitute an essential component of the innate immune response, readily killing most bacteria through phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs) among other mechanisms. These cells play an unclear role in mycobacterial infections such as Mycobacterium avium subspecies paratuberculosis (Map), the etiological agent of paratuberculosis, and its response is particularly understudied in ruminants. Herein, a wide set of techniques were adapted, or newly developed, to study the in vitro response of caprine neutrophils after Map infection. Immunofluorescence was used to demonstrate, simultaneously, chemotaxis, phagocytosis, degranulation, and NETs. The quantification of neutrophil phagocytic activity against Map at a 1:10 multiplicity of infection (MOI), through flow cytometry, showed values that varied from 4.54 to 5.63% of phagocyting neutrophils. By immunofluorescence, a 73.3 ± 14.5% of the fields showed NETs, and the mean release of DNA, attributable to NETosis, calculated through a fluorometric method, was 16.2 ± 3.5%. In addition, the RNA expression of TGF-β, TNF and IL-1β cytokines, measured through reverse transcription qPCR, was significantly higher in the two latter. Overall, neutrophil response was proportional to the number of bacteria. This work confirms that the simultaneous study of several neutrophil mechanisms, and the combination of different methodologies, are essential to reach a comprehensive understanding of neutrophil response against pathogens, demonstrates that, in vitro, caprine neutrophils display a strong innate response against Map, using their entire repertoire of effector functions, and sets the basis for further in vitro and in vivo studies on the role of neutrophils in paratuberculosis.
中性粒细胞是先天免疫反应的重要组成部分，通过吞噬作用、脱颗粒和释放中性粒细胞胞外陷阱（NETs）等机制，轻易杀死大多数细菌。这些细胞在分枝杆菌感染中扮演着不明角色，例如鸟分枝杆菌副结核亚种（Map），这是副结核病的病原体，其在反刍动物中的反应尤其研究不足。在此，我们采用了一系列广泛的技术，或开发了新技术，来研究 Map 感染后山羊中性粒细胞的体外反应。免疫荧光用于同时显示趋化性、吞噬作用、脱颗粒和 NETs。通过流式细胞术定量中性粒细胞对 Map 的吞噬活性，在 1:10 的感染倍数（MOI）下，吞噬中性粒细胞的比例为 4.54%至 5.63%。通过免疫荧光，73.3%±14.5%的视野显示有 NETs，通过荧光法计算的 NETosis 相关 DNA 释放平均值为 16.2%±3.5%。此外，通过逆转录 qPCR 测量的 TGF-β、TNF 和 IL-1β 细胞因子的 RNA 表达在后两者中显著升高。总的来说，中性粒细胞的反应与细菌数量成正比。这项工作证实了同时研究几种中性粒细胞机制，并结合不同的方法学，对于全面了解中性粒细胞对病原体的反应至关重要，表明体外山羊中性粒细胞使用其全部效应功能表现出强烈的先天反应针对 Map，并为进一步研究中性粒细胞在副结核病中的作用的体外和体内研究奠定了基础。
