In vivoanalysis ofTrypanosoma cruzipersistence foci at single cell resolution

Abstract

247 23 Text: 4892 24 25 2 ABSTRACT Infections with Trypanosoma cruzi are usually life-long despite 26 generating a strong adaptive immune response. Identifying the sites of parasite 27persistence is therefore crucial to understand how T. cruzi avoids immune-mediated 28 destruction. However, this is a major technical challenge because the parasite burden 29 during chronic infections is extremely low. Here, we describe an integrated approach 30 involving comprehensive tissue processing, ex vivo imaging, and confocal 31 microscopy, which has allowed us to visualise infected host cells in murine tissue, with 32 exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection 33 level of <20 parasites, we show that in the colon, smooth muscle myocytes in the 34 circular muscle layer are the most common infected host cell type. Typically, during 35 chronic infections, the entire colon of a mouse contains only a few hundred parasites, 36 often concentrated in a small number of cells containing >200 parasites, that we term 37 mega-nests. In contrast, during the acute stage, when the total parasite burden is 38 considerably higher and many cells are infected, nests containing >50 parasites are 39 rarely found. In C3H/HeN mice, but not BALB/c, we identified skeletal muscle as a 40 major site of persistence during the chronic stage, with most parasites found in large 41 mega-nests within the muscle fibres. Finally, we report that parasites are also 42 frequently found in the skin during chronic murine infections, often in multiple infection 43 foci. In addition to being a site of parasite persistence, this anatomical reservoir could 44 play an important role in insect-mediated transmission, and have implications for drug 45 development. 46 47 48 49 3 IMPORTANCE Trypanosoma cruzi causes Chagas disease, the most important 50 parasitic infection in Latin America. Major pathologies include severe damage to the 51 heart and digestive tract, although symptoms do not usually appear until decades after 52 infection. Research has been hampered by the complex nature of the disease and 53 technical difficulties in locating the extremely low number of parasites. Here, using 54 highly sensitive imaging technology, we reveal the sites of parasite persistence in 55 experimental mice at single-cell resolution. We show that parasites are frequently 56 located in smooth muscle cells in the circular muscle layer of the colon, and that 57 skeletal muscle cells and the skin can also be important reservoirs. This information 58 provides a framework for investigating how the parasite is able to survive as a life-long 59 infection, despite a vigorous immune response. It also informs drug-development 60 strategies by identifying tissue sites that must be accessed to achieve a curative 61 outcome.62 63 64 65 66 KEY WORDS: Trypanosoma cruzi, Chagas disease, chronic persistence, murine 67 imaging, colon, skeletal muscle, skin 68 69 70 71 72 73 74 4 INTRODUCTION The intracellular protozoan parasite Trypanosoma cruzi is the...

Challenges in Chagas Disease Drug Development

“…Visible light towards the red end of the spectrum (617 nm in this case) has greater tissue penetration due to reductions in the absorbance and scattering of the emitted light. Using these systems, chronic T. cruzi infections in mice can be monitored for more than one year, with a limit of detection close to 100 parasites using in vivo imaging [100], and less than 20 parasites when ex vivo imaging is subsequently used to examine mouse tissue [103]. With in vivo imaging, there is a linear relationship between the parasite burden (above 1000 parasites) and whole-body bioluminescence intensity [99].…”

“…parasites using in vivo imaging [100], and less than 20 parasites when ex vivo imaging is subsequently used to examine mouse tissue [103]. With in vivo imaging, there is a linear relationship between the parasite burden (above 1000 parasites) and whole-body bioluminescence intensity [99].…”

“…In this infection model, the predominant long-term sites of parasite persistence are the colon and/or stomach. Infection of other organs/tissues is more sporadic, although parasites are often located in the skin [103]. (B) Exploiting ex vivo imaging to assess drug efficacy against chronic T. cruzi infection.…”

“…During the acute stage, parasites infect all organs and tissues, whereas in the chronic stage, they are mainly confined to the colon, stomach and skin ( Figure 3 A). In C3H/HeN mice, infection of skeletal muscle is also a regular feature of the chronic stage [ 101 , 103 ]. As in humans, T. cruzi infections are generally life-long, and in vivo imaging has revealed a highly dynamic profile during the chronic stage in which transient bioluminescence foci appear and disappear over a time period of hours.…”

Host‐parasite dynamics in Chagas disease from systemic to hyper‐local scales

“…The well‐studied parasite strain CL Brener is also commonly detected in the skin of BALB/c mice 38 but only sporadically in other sites, for example skeletal muscle, lung, adipose. More virulent parasite strains (eg Brazil, Colombiana, Tulahuen, VFRA) and certain mice (eg C3H) are associated with more disseminated infections, including heart and skeletal muscle localization 29,30,170‐175 . There have been few robust data on the relevant cell types within any of these chronically infected tissues, but recent in vivo imaging analysis at single cell resolution revealed smooth muscle cells as the most frequent targets in the colon 175 …”