Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil.
Laboratório de Ciclo Celular-Center for Research on Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil.
Curr Microbiol. 2022 Jan 4;79(2):57. doi: 10.1007/s00284-021-02720-5.
Leptospirosis is a worldwide zoonosis and a serious public health threat in tropical and subtropical areas. The etiologic agents of leptospirosis are pathogenic spirochetes from the genus Leptospira. In severe cases, patients develop a pulmonary hemorrhage that is associated with high fatality rates. Several animal models were established for leptospirosis studies, such as rodents, dogs, and monkeys. Although useful to study the relationship among Leptospira and its hosts, the animal models still exhibit economic and ethical limitation reasons and do not fully represent the human infection. As an attempt to bridge the gap between animal studies and clinical information from patients, we established a three-dimensional (3-D) human lung cell culture for Leptospira infection. We show that Leptospira is able to efficiently infect the cell lung spheroids and also to infiltrate in deeper areas of the cell aggregates. The ability to infect the 3-D lung cell aggregates was time-dependent. The 3-D spheroids infection occurred up to 120 h in studies with two serovars, Canicola and Copenhageni. We standardized the number of bacteria in the initial inoculum for infection of the spheroids and we also propose two alternative culture media conditions. This new approach was validated by assessing the expression of three genes of Leptospira related to virulence and motility. The transcripts of these genes increased in both culture conditions, however, in higher rates and earlier times in the 3-D culture. We also assessed the production of chemokines by the 3-D spheroids before and after Leptospira infection, confirming induction of two of them, mainly in the 3-D spheroids. Chemokine CCL2 was expressed only in the 3-D cell culture. Increasing of this chemokine was observed previously in infected animal models. This new approach provides an opportunity to study the interaction of Leptospira with the human lung epithelium in vitro.
钩端螺旋体病是一种全球性的动物传染病，也是热带和亚热带地区严重的公共卫生威胁。钩端螺旋体病的病原体是来自钩端螺旋体属的致病性螺旋体。在严重的情况下，患者会发生肺出血，这与高死亡率有关。已经建立了几种动物模型来研究钩端螺旋体病，如啮齿动物、狗和猴子。虽然这些动物模型有助于研究钩端螺旋体与其宿主之间的关系，但它们仍然存在经济和伦理方面的限制，并且不能完全代表人类感染。为了尝试弥合动物研究与患者临床信息之间的差距，我们建立了一种用于钩端螺旋体感染的三维（3-D）人肺细胞培养。我们表明，钩端螺旋体能有效地感染细胞肺球体，并且还能渗透到细胞聚集体的更深区域。感染 3-D 肺细胞聚集体的能力是时间依赖性的。在对 Canicola 和 Copenhageni 两种血清型进行的研究中，3-D 球体感染可持续长达 120 小时。我们对初始接种物中的细菌数量进行了标准化，并且还提出了两种替代的培养条件。通过评估与毒力和运动性相关的三个钩端螺旋体基因的表达，验证了这种新方法的有效性。在这两种培养条件下，这些基因的转录本都增加了，但是在 3-D 培养中增加的速率和时间更早。我们还在钩端螺旋体感染前后评估了 3-D 球体产生的趋化因子，证实了其中两种趋化因子的诱导，主要是在 3-D 球体中。趋化因子 CCL2 仅在 3-D 细胞培养中表达。在感染动物模型中观察到了这种趋化因子的增加。这种新方法为在体外研究钩端螺旋体与人肺上皮的相互作用提供了机会。
