Treg cells in Gal-8-treated retinas are highly anti-inflammatory, inducible Treg cells.

<p>Retina-infiltrating cells were collected from uveitic mice treated with Gal-8 or vehicle at 24 d p.i. (A) Representative scatter plots show neuropilin-1 staining of Foxp3⁺ Treg cells. (B-D) Representative scatter plots (B), as well as frequency (C) and number (D) of CTLA-4, IL-10, and CD103 positive retinal Treg cells. (B-D) Retinas from five mice per group were pooled, and data are reported as fold change over control. Because of the limiting number of retinal lymphocytes, only one pooled FACS replicate was measured per group per experiment. Error bars represent mean ± SEM of two independent experiments. P values were determined by Student’s <i>t</i> test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Galectin-8 ameliorates EAU pathology.

<p>(A) B6 mice were immunized with IRBP peptide (1–20) to induce uveitis. Groups of at least 5 mice each were injected with vehicle or Gal-8 and imaged as indicated. (B) Representative fundus images of vehicle- and Gal-8-treated mice at day 0 and 24. Blue arrowhead: optic disc inflammation; white arrowhead: immune cell infiltrates; red arrowhead: engorged vasculature; green dotted line: retinal atrophy. Retinal vascular changes (C), lymphocyte infiltration to the retina (D), and overall uveitis clinical score (E) were determined for the most severe eye of each mouse and averaged across three independent experiments with at least five vehicle- and five Gal-8-treated mice each. Error bars are SEM from three independent experiments with a total of 18 vehicle- and 20 Gal-8-treated mice. P values were determined by Mann-Whitney <i>U</i> test. *, p < 0.05.</p

Galectin-8 inhibits inflammation of the retina.

<p>Retina-infiltrating cells were collected from uveitic mice treated with Gal-8 or vehicle at 24 d p.i. (A) Representative scatter plots of Foxp3, IL-4, IFNγ, and IL-17A staining are shown, and frequency and number of retinal Treg, T_H2, T_H1, and T_H17 cells are quantified. (B) Cell ratios for total numbers of Treg per T_H1 cell and Treg per T_H17 cell in the retina. (C) Cytokines in pooled retinal extracts were measured by ELISA. (A-C) Retinas from five mice per group were pooled, and data are reported as fold change over control. Because of the limiting number of retinal lymphocytes, only one pooled FACS replicate was measured per group per experiment. ELISA samples were measured in quadruplicate. Error bars represent mean ± SEM of two independent experiments. P values were determined by Student’s <i>t</i> test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.</p

Galectin-8 promotes Treg cell differentiation in the dLN during EAU.

<p>dLN and spleen cells were collected from uveitic mice treated with Gal-8 or vehicle 24 d p.i. Cells from the dLN (A) or spleen (B) were stained for Foxp3, IL-4, IFNγ, and IL-17A, and representative scatter plots gated on CD4⁺ are shown, along with frequency and total number of each T cell subset. (C) Cytokine secretion from dLN cells stimulated <i>ex vivo</i> with IRBP_1-20 for 48 h. FACS and ELISA samples were measured in triplicate. Error bars are mean ± SEM from three independent experiments with 18 vehicle- and 20 Gal-8-treated mice. P values were determined by Student’s <i>t</i> test. *, p < 0.05; ****, p < 0.0001.</p

Histopathological findings in <i>Lgals1</i>^<i>-/-</i> and WT mice at 19 dpi with <i>T</i>. <i>cruzi</i> Tulahuén strain.

<p>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. *<i>p</i><0.05. F: Female mice; M: Male mice.</p

Effect of Gal–1 on phosphatidylserine exposure in <i>T</i>. <i>cruzi</i> infected HL–1 cells.

<p>Cells were incubated with rGal–1 (10 and 50 μg/ml) for 18 h and, then infected with <i>T</i>. <i>cruzi</i>, 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. *<i>p</i><0.05; **<i>p</i><0.01. Only comparisons between infected groups were shown.</p

Effect of exogenous rGal–1 in <i>T</i>. <i>cruzi</i> infection.

<p>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 <i>T</i>. <i>cruzi</i> Tulahuén (A) or Brazil strain (D), cells were fixed and stained with an anti-<i>T</i>. <i>cruzi</i> mouse serum. Representative images are shown in (B) and (E). Similar experiments were performed after 2 dpi with <i>T</i>. <i>cruzi</i> 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-<i>T</i>. <i>cruzi</i> 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. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

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

<p>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. *<i>p</i><0.05, **<i>p</i><0.01 <i>vs</i>. WT mice; ^$<i>p</i><0.05, ^$$<i>p</i><0.01 <i>vs</i>. male mice. For survival rate, statistical analysis was achieved with Log-rank test.</p

Expression and release of Gal–1 in cultures of HL–1 cells infected with <i>T</i>. <i>cruzi</i>.

<p>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 <i>t</i> test for data shown in A (a <i>vs</i> b) and using one-way ANOVA followed by Tukey test in the remaining experiments. *<i>p</i><0.05; ***<i>p</i><0.001.</p

Binding of rGal–1 to <i>T</i>. <i>cruzi</i> trypomastigotes.

<p>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.</p