In vivo analysis of Trypanosoma cruzi persistence foci at single cell resolution
In vivo analysis of Trypanosoma cruzi persistence foci at single cell resolution
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单细胞分辨率克氏锥虫持久性病灶的体内分析
DOI：
10.1101/2020.05.13.092551
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Ward A
中科院分区：
文献类型：
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作者：
Ward A
克氏锥虫感染通常是终身的，尽管产生强烈的适应性免疫反应。因此，确定寄生虫持续存在的位点对于理解T。Cruzi避免了免疫介导的破坏。然而，这是一个重大的技术挑战，因为慢性感染期间的寄生虫负担极低。在这里，我们描述了一个综合的方法，涉及全面的组织处理，离体成像，共聚焦显微镜，这使我们能够可视化感染的宿主细胞在小鼠组织与精致的灵敏度。使用生物发光引导的组织取样，检测水平<20个寄生虫，我们表明在结肠中，环形肌层中的平滑肌肌细胞是最常见的感染宿主细胞类型。通常，在慢性感染期间，小鼠的整个结肠仅含有几百个寄生虫，通常集中在少数细胞中，每个细胞含有>200个寄生虫，我们称之为巨型巢。相反，在急性期，当总的寄生虫负荷相当高，许多细胞被感染，巢含有>50寄生虫很少found. In C3 H/HeN小鼠，但不是BALB/c小鼠，我们确定骨骼肌作为一个主要的网站的持久性在慢性期，与大多数寄生虫被发现在大的巨型巢内的肌肉纤维。最后，我们报告说，寄生虫也经常在皮肤中发现慢性鼠感染，往往在多个感染灶。除了作为寄生虫持续存在的场所外，这个解剖学上的储存库可能在昆虫介导的传播中发挥重要作用，并对药物开发产生影响。重要提示克氏锥虫导致南美锥虫病，这是拉丁美洲最重要的寄生虫感染。主要的病理包括心脏和消化道的严重损害，尽管症状通常在感染后几十年才出现。由于这种疾病的复杂性质和定位极少量寄生虫的技术困难，研究受到阻碍。在这里，使用高灵敏度的成像技术，我们揭示了寄生虫的持久性在慢性阶段感染的实验小鼠在单细胞分辨率的网站。我们发现，寄生虫经常位于平滑肌细胞在结肠的环形肌肉层，骨骼肌细胞和皮肤也可以是重要的水库。这一信息提供了一个框架，调查寄生虫是如何能够生存作为一个终身感染，尽管有强烈的免疫反应。它还通过确定必须进入以实现治愈结果的组织部位来告知药物开发策略。
Infections with Trypanosoma cruzi are usually lifelong despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understanding how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge, because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing,ex vivoimaging, and confocal microscopy, which allowed us to visualize infected host cells in murine tissue with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we showed that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells each containing >200 parasites, which we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c mice, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites being found in large mega-nests within the muscle fibers. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission and have implications for drug development.IMPORTANCETrypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence during chronic-stage infections of experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a lifelong infection, despite a vigorous immune response. It also informs drug development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.