Differential Role of TGF-β in Extracellular Matrix Regulation During Trypanosoma cruzi-Host Cell Interaction

Figures

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  Figure 1. Transforming growth factor beta (TGF-β) differentially modulates fibronectin (FN) in cardiomyocytes, skeletal myoblasts L6E9, and cardiac fibroblasts. (A) Normal cardiomyocytes showed FN fibrils on surface. The addition of 1 ng/mL of TGF-β (B) did not alter the FN expression in normal cardiomyocytes. Only 10 ng/mL of TGF-β induced the increase of FN expression (C). In contrast, L6E9 skeletal myoblasts (E) and cardiac fibroblasts (H) presented the FN matrix stimulation after treatment with 1 ng/mL TGF-β when compared to untreated cultures (D,G). The FN increase was still detected in these two cell types with addition of 10 ng/mL (F,I) of TGF-β. (J) FN staining area was measured with Image J software, showing the thickening of FN fibrils after TGF-β treatment. * p ≤ 0.05 compared to untreated cardiomyocytes (CM); ** p ≤ 0.05 compared to untreated L6E9; # p ≤ 0.05 compared to untreated cardiac fibroblasts (CF). Bar = 20 μm.

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  Figure 2. Phosphorylated SMAD2 and p38 MAPK detection in normal cultures. (A) Comparison of SMAD2 phosphorylation (PS2) detection of CM, L6E9 skeletal myoblasts, and CF treated with TGF-β (1–10 ng/mL) per 1 h. PS2 basal levels in L6E9 skeletal myoblasts and CF were significantly greater than CM. After the addition of TGF-β, PS2 remained significantly higher in L6E9 and CF relative to CM. L6E9 skeletal myoblasts responded to TGF-β stimulus in a dose dependent manner. CF showed a slight level of PS2 increase. * p ≤ 0.05 compared to CM; ** p ≤ 0.05 compared with CM in a different concentration; # p ≤ 0.05 compared with L6E9 control. N = 3; (B) Comparison of phosho-p38 MAPK detection of CM, L6E9 skeletal myoblasts, and CF treated with TGF-β (1–10ng/mL) per 1 h. L6E9 skeletal myoblasts presented a high level of p38 MAPK phosphorylation when compared with CM. When CM was compared with CF, a higher p38 MAPK phosphorylation after stimulation with TGF-β (1–10 ng/mL) was observed. * p ≤ 0.05 compared to CM and ** p ≤ 0.05 compared with CM in a different concentration. N = 3.

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  Figure 3. Fibronectin expression in L6E9 skeletal myoblasts and cardiac fibroblasts treated with TGF-β. (A) In normal cultures, treatment with 1 ng/mL TGF-β for 48 h triggered the increase of FN expression in L6E9 skeletal myoblasts in a dose-dependent manner after the addition of 1 and 10 ng/mL TGF-β. In L6E9 skeletal myoblasts infected with T. cruzi (72 h) and treated with TGF-β (1 and 10 ng/mL) an increase of FN expression was observed when compared with the infected and untreated cultures, but there was a reduction in the FN expression when compared with the uninfected cultures, even after treatment with TGF-β (1 and 10 ng/mL). * p ≤ 0.05 compared to uninfected pairs; # p ≤ 0.05 compared to untreated control and uninfected culture; N = 4. (B) Normal CF was treated with TGF-β (1 and 10 ng/mL) and showed an increase of FN expression according to the dose administered. In CF infected and untreated, there was an increase in the FN expression when compared with their normal counterparts. In CF infected and treated with TGF-β (1 and 10 ng/mL), a reduction of FN expression was observed when compared with their treated pairs. Pre-treatment of the culture with p38 (SB203580) and SMAD2…

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  Figure 4. Distribution of fibronectin in L6E9 skeletal myoblasts infected with T. cruzi and treated with TGF-β. T. cruzi infection (72 h) reduced FN fibrils staining (red) in L6E9 skeletal myoblasts (B,C) when compared to uninfected controls (A). The treatment of T. cruzi infected L6E9 skeletal myoblasts with 1 ng/mL (E,F) or 10 ng/mL (H,I) of TGF-β induced an increase in FN expression only in uninfected areas in this culture when compared to uninfected TGF-β treated cultures ((D) 1 ng/mL; (G) 10 ng/mL), which presented widespread enhancement of FN. (J) Measurement of FN staining area with Image J software showed a reduction of the FN area after infection and a thickening of FN fibrils after TGF-β treatment in uninfected cultures. Differential interference contrast (DIC) was used to show the intracellular parasites in the host cells. * p ≤ 0.05 vs. untreated, uninfected control (C72h); ** p ≤ 0.05 vs. uninfected + 1 ng/mL TGF-β; # p ≤ 0.05 vs. uninfected + 10 ng/mL TGF-β. Bar = 20 μm.

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  Figure 5. Distribution of fibronectin in cardiac fibroblasts infected with T. cruzi and treated with TGF-β. FN staining (red) in untreated T. cruzi infected CF (B,C) showed a general intensification of FN expression after 72 h of interaction, while highly infected cells presented a localized reduction when compared to uninfected controls (A). CF cultures showed higher FN expression after treatment with 1 ng/mL (D) and 10 ng/mL (G) of TGF-β. CF still displayed a localized reduction of FN staining in infected cells, even after TGF-β treatment ((E,F) 1 ng/mL; (H,I) 10 ng/mL). (J) Quantification of FN fibril thickness by image processing with Image J software. DAPI (blue) was used to label the nucleus of the host cell and the kinetoplast of intracellular parasites. * p ≤ 0.05 vs. untreated, uninfected control (C72h); ** p ≤ 0.05 vs. uninfected + 1 ng/mL TGF-β; # p ≤ 0.05 vs. uninfected + 10 ng/mL TGF-β. Bar = 20 μm.

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  Figure 6. PS2, phospho-p38 MAPK, and phospho-c-Jun detection in normal and infected cardiac fibroblasts with T. cruzi. (A) PS2 detection. Normal CF showed an increase of SMAD2 phosphorylation when treated with 10 ng/mL of TGF-β. Infected CF were unable to trigger the classical signaling pathway, even when treated with 10 ng/mL, compared to the uninfected pair. The SB431542 inhibitor prevented SMAD2 phosphorylation in CF cultures after stimulation with TGF-β. (B) Phosphorylated p38 MAPK detection. In normal CF treated with 10 ng/mL of TGF-β, an increase of p38 MAPK phosphorylation detection was observed. T. cruzi infection did not result in significant differences in p38 MAPK phosphorylation, although there was a tendency to increase in this signaling pathway. After addition of 10 ng/mL of TGF-β, the signaling pathway was triggered in CF. The inhibitor SB431542 prevented the p38 MAPK phosphorylation in both normal and infected CF treated with 10 ng/mL TGF-β. (C) Phosphorylated c-Jun detection. Note that infection by T. cruzi led to an increase in the c-Jun signaling pathway. The addition of TGF-β in normal cultures resulted in a 25% increase in c-Jun detection. The increase triggered by the…

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  Figure 7. Phosphorylated SMAD2 detection in Trypanosoma cruzi infected cardiac fibroblasts. PS2 staining is represented in red (A,C,E,G,I,K,M,O), while cell nuclei are depicted in blue after DAPI staining together with DIC (B,D,F,H,J,L,N,P). (A,B) Control, uninfected cultures (C72h), showing constitutive PS2 expression; (C,D) T. cruzi infection (72h- Y72h) reduced PS2 detection (Y72h); (E,F) TGF-β treatment stimulated PS2 in uninfected cultures; (G,H) Infection with T. cruzi prevented PS2 signaling in CF; SB431542 inhibited PS2 detection in uninfected (I,J) and T. cruzi infected (K,L) CF cultures; (M,N) SB431542 prevented TGF-β stimulation of PS2; (O,P) T. cruzi infection and SB431542 treatment resulted in low detection of PS2 in CF. (Q) Quantification of percentage of nuclei positive for PS2. * p ≤ 0.05 vs. untreated, uninfected control; ** p ≤ 0.05 vs. C72h + 10 ng/mL TGF-β. Bar = 10 μm.

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  Figure 8. Cardiac fibroblast proliferation treated with TGF-β. Cardiac fibroblasts treated with different concentrations of TGF-β (1 and 10 ng/mL) showed a high proliferation in a dose dependent manner when compared with CF untreated. * p ≤ 0.05 vs. untreated, uninfected control; ** p ≤ 0.05 vs. control + 1 ng/mL TGF-β; # p ≤ 0.05 vs. T. cruzi 72 h + 1 ng/mL TGF-β. N = 3.