Adipose Tissue-Derived Mesenchymal Stromal Cells Protect Mice Infected with <i>Trypanosoma cruzi</i> from Cardiac Damage through Modulation of Anti-parasite Immunity

<div><p>Background</p><p>Chagas disease, caused by the protozoan <i>Trypanosoma cruzi</i> (<i>T</i>.<i>cruzi</i>), is a complex disease endemic in Central and South America. It has been gathering interest due to increases in non-vectorial forms of transmission, especially in developed countries. The objective of this work was to investigate if adipose tissue-derived mesenchymal stromal cells (ASC) can alter the course of the disease and attenuate pathology in a mouse model of chagasic cardiomyopathy.</p><p>Methodology/Principal Findings</p><p>ASC were injected intraperitoneally at 3 days post-infection (dpi). Tracking by bioluminescence showed that cells remained in the abdominal cavity for up to 9 days after injection and most of them migrated to the abdominal or subcutaneous fat, an early parasite reservoir. ASC injection resulted in a significant reduction in blood parasitemia, which was followed by a decrease in cardiac tissue inflammation, parasitism and fibrosis at 30 dpi. At the same time point, analyses of cytokine release in cells isolated from the heart and exposed to <i>T</i>. <i>cruzi</i> antigens indicated an anti-inflammatory response in ASC-treated animals. In parallel, splenocytes exposed to the same antigens produced a pro-inflammatory response, which is important for the control of parasite replication, in placebo and ASC-treated groups. However, splenocytes from the ASC group released higher levels of IL-10. At 60 dpi, magnetic resonance imaging revealed that right ventricular (RV) dilation was prevented in ASC-treated mice.</p><p>Conclusions/Significance</p><p>In conclusion, the injection of ASC early after <i>T</i>. <i>cruzi</i> infection prevents RV remodeling through the modulation of immune responses. Lymphoid organ response to the parasite promoted the control of parasite burden, while the heart, a target organ of Chagas disease, was protected from damage due to an improved control of inflammation in ASC-treated mice.</p></div

Cytokine release in non-infected and infected mice in response to <i>T</i>. <i>cruzi</i> antigens (tAg).

<p>(A) Splenocytes from infected animals released significantly higher levels of IFN-γ and TGF-β when compared to non-infected controls. No differences were found between placebo and ASC-treated groups in IFN-γ, TNF-α and TGF-β. IL-2 levels were increased in ASC-treated samples when compared to non-infected controls. IL-6 and IL-10 were higher in the ASC group when compared to placebo and non-infected groups. (B) When stimulated with tAg, cells derived from hearts treated with ASC released less IFN-γ and more IL-10 in comparison to placebo-treated animals. Levels of TNF-α, TGF-β and IL-6 were significantly increased in the placebo group when compared to non-infected controls, while the ASC group had intermediate levels of these cytokines. IL-2 release was low and similar between the three experimental groups (*p<0.05, numbers of samples in each group were at least non-infec: 4, placebo: 5, ASC: 5).</p

Parasitism, inflammation and fibrosis in infected mice.

<p>(A) Number of parasites (x10⁵) per mL of blood as days after infection progress. The area under the parasitemia curve was calculated for each animal and plotted in (B). ASC-treated animals (n = 14) had a significantly lower AUC when compared to placebo (n = 11) (**p = 0.0022). (C-G) H&E and (H-J) Sirius Red staining of heart tissue sections. (C) Non-infected animals showed normal myocardial fibers. Infection with <i>T</i>. <i>cruzi</i> caused a significant increase in the number of mononuclear inflammatory foci (arrows) (D) and amastigote nests (arrow heads) (F). Therapy with ASC prevented myocardial inflammation (E) and reduced the number of amastigote nests in the heart (G). Additionally, fibrosis was also increased in infected animals treated with placebo (I) when compared to non-infected mice (H), as shown by collagen fibers stained in red. Treatment with ASC reduced fibrosis in infected animals (J). Quantification of inflammation (K) (n = 7, ***p = 0.0002), amastigote nests (L) (n = 3, *p = 0.015) and fibrosis (M) (n = 3, **p = 0.0083) compared to Infec+ASC, Student’s t test.</p

Magnetic resonance imaging of chagasic mice.

<p>(A) Representative magnetic resonance images in diastole. A sequence of images from the same animal is shown in each line. Observe the enlargement of the right ventricle (RV) in the placebo group. (B) RV end-diastolic volume (EDV) was significantly higher in placebo-treated when compared to ASC-treated animals. (C) RV end-systolic volume (ESV) was significantly higher in placebo-treated when compared to ASC-treated and non-infected mice. No differences were found in RV ejection fraction (EF) (D) or in left ventricular EDV (E), ESV (F) or EF (G) (*p<0.05, non-infec n = 3, placebo n = 6, ASC n = 6 for all parameters).</p

Characterization of ASC.

<p>(A) ASC were adherent to culture flaks and exhibited a fibroblast-like spindle-shaped morphology. (B) Macroscopic view of CFU-F assay showing colonies of ASC stained by Giemsa. (C) Microscopic view exemplifying one colony of ASC also stained by Giemsa. Growth of ASC in culture was exponential (D) and the curves were converted to logarithmic scale (E) to calculate population doubling time (PDT) (n = 4 in all experiments). (F-K) Histograms showing the immunophenotype of ASC by flow cytometry. (F) Dead cells were excluded from the analysis by DAPI staining. (G-K) Dashed lines represent isotype controls and the percentage of positive events is shown on the right upper quadrant. Cells were predominantly positive for CD90.2 (G) and Sca-1 (H). Expression of CD105 (I) and CD73 (J) was lower. No hematopoietic contaminants were observed since cells were negative for CD45 (K). (L-O) Differentiation of ASC. (L and N) Negative controls. Cells were cultured for 21 days in standard expansion culture medium. (M) ASC cultured in adipogenic conditions exhibited lipid vacuoles in their cytoplasm shown by Oil Red O staining (white arrows). (O) Cells cultured in osteogenic conditions exhibited calcium deposits in the extracellular matrix stained by Alizarin Red. Inserts show higher magnification images of M and O.</p

Cell tracking by bioluminescence.

<p>(A) Evaluation of luminescent signal intensity in ASC transduced with the Luciferase 2 gene <i>in vitro</i>. Numbers shown below the wells indicate the amount of cells. (B) The signal increased with cell numbers in a linear fashion (R² = 0.9914). <i>In vivo</i> images (C) and quantification (D) (n = 3) of the luminescent signal showed that cells remained in the abdominal region and that there was a progressive decrease in radiance with time. The signal disappeared within 9 days of cell injection. <i>Ex vivo</i> images (E) and quantification (F) (n = 3 per day) of the luminescent signal demonstrated that the majority of the cells migrated to the abdominal or subcutaneous fat. The signal was lower in other abdominal organs, such as the spleen and liver, and virtually absent in thoracic organs such as the heart and lungs. Quantification data in D and F are shown in logarithmic scale.</p