Characterising the Immune Response Against T. cruzi in the GI tract and a Possible Role for Anti-inflammatory Cytokines TGF-b and IL-10

The recent paper Francisco et.al, (2015) identifies through in vivo imaging the GI, specifically the colon and stomach, as key sites in which T. cruzi replication is active at the chronic stage even in the presence of chemotherapeutic posacoazole treatment. The replication in this model in spite of drug treatment mirrors the types of drug failure seen in the clinic in patients with Chagas disease.

This observation links with our classical immunological understanding of the intestinal tissue as a site set up in early development to be tolerogenic. This project proposal has several aims:

1. Defining the specific tissue/cell type harbouring the parasite
2. Establishing a role for TGF-b in T. cruzi persistence

The first aim will allow characterisation of the permanent cellular reservoir of the parasite which can be inferred to intermittently release infected immune cells, that lead to the transient infection other tissues including the heart, where much of the clinical disease pathology is observed. Identification of this infected cell type would allow better targeting of future drug candidates with the goal being global sterile cure.

The phenotype of the immune response to T. cruzi has been investigated with conflicting conclusions drawn. It is widely accepted that a Th1 response, particularly high expression of IFN-y and TNF-a is associated with control of the intracellular parasite in vertebrate hosts. What is less clear is the role of the anti-inflammatory cytokine TGF-b.
Studies by Ferrao et.al, (2015) identifying TGF-b as a major determinant of successful T. cruzi host cell entry and subsequent amastigote replication. The colon, specifically identified in your Fransico et.al, (2015) paper as a site of parasite survival is well known to have a very TGF-b rich tolerogenic environment to prevent immunopathology. This proposal seeks to show whether organs in the GI tract provide an advantage sustaining T. cruzi parasite replication during posacoazole clearance of the other mouse tissues in the model.

Aims

1 - Further characterising the T. cruzi localisation of infection in the posacoazole treated mouse model

-Formalin fixed and paraffin embedded sections of parasite burdened organ stained with eosin and haematoxylin. 400x light microscope search for amastigote nests imaged previously by Roffe et al. (2012).
-T. cruzi with GFP gene insertion and expression. GI tract tissue removed from chronically infected mouse model. Tissue homogenised and put through a FACS sorter. mAb against cell type surface markers to identify cells harbouring the GFP T. cruzi.

2 - Identifying the role of anti-inflammatory cytokine TGF-b

-Inject anti TGF-b Abs at progressively increasing concentrations until a difference between treated and non-treated posacoazole mouse model is observed. Inhibition of TGF-b is observed in a similar experiment by (Vitsky et.al, 2009). The highest dose reported in this paper 50mg/kg injected clearly yielded a good inhibition of TGF-b activity as demonstrated by the dysregulation in the oral and oesophageal tissues in the mice. In the current proposal I would run the posacoazole + immunosuppression experiment with a very low 0.1mg/kg dose of the anti-TGF-b mAb and rerun the experiment with gradually increasing dosages until a significant statistical difference between the control and mAb treatment groups became apparent.