Animal models for exploring Chagas disease pathogenesis and supporting drug discovery

Abstract

SUMMARYInfections with the parasitic protozoan Trypanosoma cruzi cause Chagas disease, which results in serious cardiac and/or digestive pathology in 30%-40% of individuals. However, symptomatic disease can take decades to become apparent, and there is a broad spectrum of possible outcomes. The complex and long-term nature of this infection places a major constraint on the scope for experimental studies in humans. Accordingly, predictive animal models have been a mainstay of Chagas disease research. The resulting data have made major contributions to our understanding of parasite biology, immune responses, and disease pathogenesis and have provided a platform that informs and facilitates the global drug discovery effort. Here, we provide an overview of available animal models and illustrate how they have had a key impact across the field.

Keywords: Chagas disease; Trypanosoma cruzi; animal models; drug development; pathogenesis.

Fig 1

Mammalian stages of the Trypanosoma cruzi life cycle. Flagellated metacyclic trypomastigotes are transmitted by triatomine bugs and infect mammalian cells where they differentiate into replicative amastigotes that divide by binary fission. After 4–7 days, the parasites transform into the non-replicative bloodstream-form trypomastigote stage, escape from the host cell, and continue to propagate the infection. In the example shown, MA104 cells (an African Green monkey fetal kidney cell line) were infected with T. cruzi CL Brener parasites (DTU TcVI) expressing mScarlet fluorescence (red). mScarlet is co-expressed with luciferase as part of a bioluminescence:fluorescence fusion protein reporter encoded by an engineered gene stably integrated into a ribosomal RNA expression site (14). DNA (host and parasite) is stained with Hoechst (blue). White scale bars = 10 µM. The metacyclic trypomastigote (left) has been enlarged for visual effect.

Fig 2

Using bioluminescent/fluorescent imaging to monitor murine acute-stage T. cruzi infections. BALB/c mice were infected with the T. cruzi (CL Brener strain) that had been genetically modified to express a fusion protein that is both bioluminescent and fluorescent (see Fig. 3) (14). (A) Ex vivo bioluminescence imaging reveals the widespread dissemination of T. cruzi in mouse tissue and organs at the peak of the acute stage of infection (day 14). The top left panel is adapted from reference (105) (published under a Creative Commons license). (B) Fluorescence imaging of various tissue sections obtained from acutely infected mice [the cardiac muscle and bladder images are reprinted from reference (106)]. With the exception of skeletal muscle, host cell DNA is pseudo-colored red 4′,6-diamidino-2-phenylindole (DAPI) , and parasites are green (or yellow when imaged on a red background). In the skeletal muscle image, DNA is stained blue (also DAPI), with actin stained red (mouse mAb; Thermo Fisher MA5-12542), giving the typical striated pattern. Scale bars = 20 µm

Fig 3

Single-cell resolution imaging of a chronic T. cruzi infection focus in the mouse colon. (A) Organization of the bioluminescent:fluorescent fusion gene, following integration into a T. cruzi ribosomal locus (14). The red-shifted (R-S) luciferase, linker (black), and mNeonGreen (mNeonG) sequences are indicated. (B) Ex vivo bioluminescence image of an external colonic wall layer from a chronically infected C3H/HeN mouse imaged using the IVIS Spectrum system (Caliper Life Science). (C) Detection of fluorescent parasites in the tissue using a Zeiss LSM880 confocal microscope (Ex506 nm, Em517 nm) after bioluminescence-guided imaging. (D) Serial z-stack sections of the tissue encompassing the infected cell can be used to generate a 3D image, with parasite numbers then determined on the basis of DNA staining (DAPI, blue). White arrows indicate each parasite [adapted from reference (104)].

Fig 4

The development of cardiac fibrosis in T. cruzi-infected mice can be blocked when curative BNZ treatment is initiated in the acute stage, but not the chronic stage. BALB/c mice infected with T. cruzi (CL Brener strain) were subject to curative BNZ treatment (20 days, 100 mg/kg). (A) Quantification of collagen content (Masson’s trichome stain) in cardiac sections as a marker of fibrosis. Data are from mice where treatment was initiated 14, 22, 66, or 100 DPI (176), and the cardiac tissue was then harvested 169 DPI. (B) Micrographs highlighting the extent of cardiac fibrosis (collagen deposition—blue) in control mice and infected mice where BNZ treatment was initiated 22 or 110 DPI.

Fig 5

Tissue distribution of T. cruzi during chronic murine infections revealed by ex vivo bioluminescence imaging. BALB/c mice were infected with T. cruzi CL Brener (DTU TcVI), and C3H/HeN mice were infected with T. cruzi JR (DTU TcI), as indicated. The leftmost panel is adapted from reference (105) (published under a Creative Commons license). (A, B) When infections had progressed to the chronic stage (>100 days), tissues and organs were arranged as shown and examined by ex vivo bioluminescence imaging (104). (C) Ex vivo imaging of skin, fur side down and with adipose tissue removed. Upper image: BALB/c, Tc CL Brener. Lower image: C3H/HeN, Tc JR. Parasite strains express a red-shifted luciferase gene that was integrated into a T. cruzi ribosomal locus (101).

Fig 6

Visualizing the impact of T. cruzi infection on enteric neurons. (A) Compressed z-stack fluorescence image of a colon tissue whole mount from a C3H/HeN mouse 42 DPI with a green fluorescent T. cruzi JR reporter strain. Expanded panels (right) show 4-µm sliced single z-stack images of parasites (green) in close proximity to nerves (red; anti- Tuj1) in the region highlighted by the white arrow. Host cell DNA, gray. (B) Compressed z-stack whole-mount immunofluorescence images of colon tissue neuronal cell bodies in the myenteric nerve plexus of a C3H/HeN mouse 336 DPI with T. cruzi JR (anti-Hu; magenta). The diffuse cell morphology (lower image) illustrates the progressive deterioration of the enteric nervous system during chronic-stage DCD.

Fig 7

Investigating Chagas disease drug efficacy using bioluminescence imaging. (A) BALB/c mice chronically infected with T. cruzi CL Brener were treated orally with 30 mg/kg BNZ for either 5 or 20 days. Ventral and dorsal in vivo images of two mice per drug regimen are shown. One hundred and seventeen days post-infection, both mice that were treated for 5 days were designated as non-cured and euthanized (yellow dots). Mice given with the longer dosing regimen were further treated with cyclophosphamide (injected with 200 mg/kg on 136, 140, and 144 DPI) to promote the outgrowth of any remaining parasites. (B) Ex vivo images of organs and tissues from the mice treated with BNZ for 20 days (176 DPI). Mice that are bioluminescence negative by both in vivo and ex vivo imaging are designated as cured. The top panel is adapted from reference (105) (published under a Creative Commons license).