Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

Abstract

Background: Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection.

Methodology and principal findings: Here we investigated the contribution of galectin-1 (Gal-1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL-1 cardiac cells to Gal-1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal-1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL-1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal-1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal-1 to the cell surface. Consistent with these data, Gal-1 deficient (Lgals1-/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain.

Conclusion/significance: Our results indicate that Gal-1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions.

Fig 1. Gal–1 concentration in sera from patients with chronic Chagas disease and non-infected individuals.

Serum Gal–1 levels were determined by ELISA, as indicated in the Methods section. Statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s multiple comparison test. **p<0.01; ***p<0.001.

Fig 2. Expression and release of Gal–1 in cultures of HL–1 cells infected with T. cruzi.

Cells were infected with trypomastigotes of Tulahuén or Brazil strains, in a parasite:cell ratio of 5:1, and incubated for additional 2 or 5 days. A) Immunoblot analysis of Gal–1 expression in lysates from non-infected (a) and infected (b) HL–1 cells. Immunoreactive protein bands were semiquantified by densitometry. Results are expressed as Arbitrary Units (AU) relative to β-actin. B) RT-qPCR analysis of Gal–1 mRNA expression of non-infected and infected HL–1 cells. Results are expressed as relative to GAPDH mRNA. C) Detection of Gal–1 in the supernatant of non-infected and infected HL–1 using trypomastigotes of the Tulahuén and Brazil strains, as measured by ELISA. D) Detection of LDH activity in the supernatants of non-infected and infected HL–1 cells by using the LDH-UP kit (Weiner Lab, Argentina), following the manufacturer’s instructions. Results are expressed as Units/ml (U/ml). Data represent the mean ± SEM of three (A and B) and two (C and D) independent experiments. Statistical analysis was performed using Student’s t test for data shown in A (a vs b) and using one-way ANOVA followed by Tukey test in the remaining experiments. *p<0.05; ***p<0.001.

Fig 3. Effect of exogenous rGal–1 in T. cruzi infection.

HL–1 cells were incubated with rGal–1 (10 and 50 μg/ml) for 24 h and then infected with trypomastigotes of both strains. After 4 dpi with T. cruzi Tulahuén (A) or Brazil strain (D), cells were fixed and stained with an anti-T. cruzi mouse serum. Representative images are shown in (B) and (E). Similar experiments were performed after 2 dpi with T. cruzi of the Tulahuén (C) or Brazil strains (F). In this case, some wells were treated with 100 mM lactose, added simultaneously with rGal–1. G) HL–1 cells transfected with pcDNA3-Gal–1 vector or empty vector (mock) were infected with trypomastigotes of both strains, in the presence or absence of 100 mM lactose. Cells were fixed and stained after 2 dpi, with an anti-T. cruzi mouse serum. In all cases, the percentage of infected cells was determined by counting an average of 3,500 cells in each slide on 3–5 distinct coverslips in randomly selected fields. Results are expressed as mean ± SEM of triplicates determinations from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *p<0.05; **p<0.01; ***p<0.001.

Fig 4. Binding of rGal–1 to T. cruzi trypomastigotes.

A) Fluorescence assay of trypomastigotes incubated with rGal–1 (25 μg/ml) for 1 h, followed by incubation with a mouse anti-Gal–1 Ab labeled with Alexa Fluor 488. Staining with a rabbit polyclonal serum anti-Tc13, a surface protein presented in trypomastigotes, was used as positive control. B) Representative histograms of trypomastigotes of the Tulahuén or Brazil strain incubated with Gal-1-FITC (25 μg/ml). Red lines correspond to parasites treated with Gal-1-FITC, black lines to parasites incubated with streptavidin-FITC used as negative control.

Fig 5. Effect of Gal–1 on phosphatidylserine exposure in T. cruzi infected HL–1 cells.

Cells were incubated with rGal–1 (10 and 50 μg/ml) for 18 h and, then infected with T. cruzi, Tulahuén (A) or Brazil (B) strains. Annexin V assay was performed at 3 dpi. Results expressed as mean ± SEM are representative of two independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *p<0.05; **p<0.01. Only comparisons between infected groups were shown.

Fig 6. Glycophenotypic analysis of HL–1 cells infected with T. cruzi trypomastigotes.

Non-infected and infected HL–1 cells at 5 dpi with T. cruzi Tulahuén and Brazil strain, were incubated with different biotinylated lectins (see S1 Table), followed by incubation with FITC-streptavidin. After fixation, cells were analyzed by flow cytometry (A). Results are expressed as relative SFI, calculated as SFI of infected HL–1 cells/SFI of non-infected HL–1 cells for each lectins tested. SFI for each lectin was calculated as the ratio of the mean fluorescence of each samples stained with biotinylated lectin plus streptavidin-FITC over the mean fluorescence of the samples incubated with streptavidin-FITC alone. Dot lines show the mean value of relative SFI of infected over non-infected HL–1 cells incubated with streptavidin-FITC (baseline). Relative percentage of positive cells (infected/non-infected) for SNA staining is shown in B). Results are expressed as mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett´s test. *p<0.05; **p<0.01. Glycophenotypic analysis of infected HL–1 cells was also determined by fluorescence assay. Representative images of LEL and PHA-L staining are shown in C). Binding curve of rGal–1 to HL–1 cells infected with T. cruzi trypomastigotes of the Tulahuén strain (D). Non-infected and infected cells at 5 dpi with T. cruzi Tulahuén strain, were incubated with different concentration of rGal–1, in the absence or presence of lactose (100 mM) and then revealed with FITC labeled anti-mouse Gal–1 Ab. After 30 min, cells were fixed and a minimum of 10,000 events were acquired on a FACSAria flow cytometer. Nonspecific binding was determined with FITC conjugated streptavidin alone.

Fig 7. Parasitemia levels (A) and survival rate (B) of WT and Lgals1 ^-/- mice acutely infected with T. cruzi Tulahuén strain, via the intraperitoneal route.

For parasitemia levels, each point represents the mean ± SEM of 5–15 animals per group, and statistical analysis was performed using Mann-Whitney U test. *p<0.05, **p<0.01 vs. WT mice; ^$ p<0.05, ^$$ p<0.01 vs. male mice. For survival rate, statistical analysis was achieved with Log-rank test.

Fig 8. Histopathological findings in Lgals1 ^-/- and WT mice at 19 dpi with T. cruzi Tulahuén strain.

A) Microphotographs representative of heart and skeletal muscle histopathological abnormalities (H&E). Parasite density (B) and Inflammation Index (C) were calculated as indicated in the Methods section. Bars represent mean ± SEM of 5–7 mice per group. Statistical analysis was performed using Mann-Whitney U test. *p<0.05. F: Female mice; M: Male mice.