Antigen-Based Nano-Immunotherapy Controls Parasite Persistence, Inflammatory and Oxidative Stress, and Cardiac Fibrosis, the Hallmarks of Chronic Chagas Cardiomyopathy, in A Mouse Model of Trypanosoma cruzi Infection

Abstract

Chagas cardiomyopathy is caused by Trypanosoma cruzi (Tc). We identified two candidate antigens (TcG2 and TcG4) that elicit antibodies and T cell responses in naturally infected diverse hosts. In this study, we cloned TcG2 and TcG4 in a nanovector and evaluated whether nano-immunotherapy (referred as nano2/4) offers resistance to chronic Chagas disease. For this, C57BL/6 mice were infected with Tc and given nano2/4 at 21 and 42 days post-infection (pi). Non-infected, infected, and infected mice treated with pcDNA3.1 expression plasmid encoding TcG2/TcG4 (referred as p2/4) were used as controls. All mice responded to Tc infection with expansion and functional activation of splenic lymphocytes. Flow cytometry showed that frequency of splenic, poly-functional CD4 + and CD8 + T cells expressing interferon-γ, perforin, and granzyme B were increased by immunotherapy (Tc.nano2/4 > Tc.p2/4) and associated with 88%-99.7% decline in cardiac and skeletal (SK) tissue levels of parasite burden (Tc.nano2/4 > Tc.p2/4) in Chagas mice. Subsequently, Tc.nano2/4 mice exhibited a significant decline in peripheral and tissues levels of oxidative stress (e.g., 4-hydroxynonenal, protein carbonyls) and inflammatory infiltrate that otherwise were pronounced in Chagas mice. Further, nano2/4 therapy was effective in controlling the tissue infiltration of pro-fibrotic macrophages and established a balanced environment controlling the expression of collagens, metalloproteinases, and other markers of cardiomyopathy and improving the expression of Myh7 (encodes β myosin heavy chain) and Gsk3b (encodes glycogen synthase kinase 3) required for maintaining cardiac contractility in Chagas heart. We conclude that nano2/4 enhances the systemic T cell immunity that improves the host's ability to control chronic parasite persistence and Chagas cardiomyopathy.

Roles of MicroRNA-122 in Cardiovascular Fibrosis and Related Diseases

“…A variety of alterations in the interstitial myocardial collagen network, macrophages, CMs, CFs, ECs and vascular cells are closely linked with fibrillar alteration in the heart. At the molecular and cellular levels, cardiovascular fibrosis is linked with reduced nitric oxide release, increased ROS generation, the activation of transcription factors, the stimulation of proinflammatory and profibrotic pathways, increased collagen deposition and ECM remodeling [1,3,[28][29][30][31]. Inflammation and oxidative stress seem to be positively associated with remodeling processes, ultimately contributing to cardiac dilation and dysfunction [13,29].…”

“…Inflammation and oxidative stress seem to be positively associated with remodeling processes, ultimately contributing to cardiac dilation and dysfunction [ 13 , 29 ]. MiRs participate in regulating cell proliferation, migration, differentiation, and apoptosis and function as crucial determinants and biomarkers of cardiovascular fibrosis [ 1 , 5 , 6 , 30 , 32 ]. In particular, miR-122 has been implicated in the progression of fibrosis and acts as a circulatory biomarker in hypertension and HF [ 1 , 6 , 17 , 18 , 33 – 37 ].…”

Table_1.docx

“…The fulllength cDNAs for the two antigens were previously cloned into pCDNA3.1 eukaryotic expression plasmid and the recombinant plasmids were purified by anion exchange chromatography by using a Qiagen Endo-free maxi prep kit (Qiagen, Chatsworth, CA) (9, 10). A CMV promoter-based NTC9385R-MCS nanoplasmid (Nature technology, Lincoln, NE) was utilized for sub-cloning the full-length genes encoding for TcG2 and TcG4 (18). The recombinant nanoplasmids NTC9385R-TcG2 and NTC9385R-TcG4 were sequenced to confirm the orientation and open reading frame, and purified by using the HyperGRO fermentation process (18).…”

“…A CMV promoter-based NTC9385R-MCS nanoplasmid (Nature technology, Lincoln, NE) was utilized for sub-cloning the full-length genes encoding for TcG2 and TcG4 (18). The recombinant nanoplasmids NTC9385R-TcG2 and NTC9385R-TcG4 were sequenced to confirm the orientation and open reading frame, and purified by using the HyperGRO fermentation process (18).…”

“…As controls, tubes containing unstained cells and cells incubated with isotype matched IgG, live/dead stain and FMO controls were included. Data were acquired and analyzed by using a FlowJo software (v.10.5.3; TreeStar, San Carlo, CA) (18). All antibodies are listed in Table S1.…”