Protective Effect of Galectin-1 during Histoplasma capsulatum Infection Is Associated with Prostaglandin E2 and Nitric Oxide Modulation

Abstract

Histoplasma capsulatum is a dimorphic fungus that develops a yeast-like morphology in host's tissue, responsible for the pulmonary disease histoplasmosis. The recent increase in the incidence of histoplasmosis in immunocompromised patients highlights the need of understanding immunological controls of fungal infections. Here, we describe our discovery of the role of endogenous galectin-1 (Gal-1) in the immune pathophysiology of experimental histoplasmosis. All infected wild-type (WT) mice survived while only 1/3 of Lgals1^-/- mice genetically deficient in Gal-1 survived 30 days after infection. Although infected Lgals1^-/- mice had increased proinflammatory cytokines, nitric oxide (NO), and elevations in neutrophil pulmonary infiltration, they presented higher fungal load in lungs and spleen. Infected lung and infected macrophages from Lgals1^-/- mice exhibited elevated levels of prostaglandin E₂ (PGE₂, a prostanoid regulator of macrophage activation) and prostaglandin E synthase 2 (Ptgs2) mRNA. Gal-1 did not bind to cell surface of yeast phase of H. capsulatum, in vitro, suggesting that Gal-1 contributed to phagocytes response to infection rather than directly killing the yeast. The data provides the first demonstration of endogenous Gal-1 in the protective immune response against H. capsulatum associated with NO and PGE₂ as an important lipid mediator in the pathogenesis of histoplasmosis.

Figure 1

Gal-1 knockout mice are sensitive to H. capsulatum infection. WT and Lgals1^−/− mice were infected i.t. with 5 × 10⁵ viable H. capsulatum yeast (sublethal inoculum) and survival was monitored for 30 days. Control mice (uninfected) received i.t. 100 μL PBS (data not shown). Data are representative of one of the two experiments performed independently (n = 10 per group) and Mantel-Cox log-rank (χ ² “chi-squared”) was used. ^# p < 0.05 WT versus Lgals1^−/−, both infected.

Figure 2

Lgals1^−/− mice have a higher yeast burden in the lung and spleen day 15 after infection. On day 15 after infection with H. capsulatum (5 × 10⁵ viable yeasts), animals were sacrificed and tissue samples were harvested. (a) Lung sections (5 μm) from WT and Lgals1^−/− mice were stained with silver (Grocott's methanamine silver (GMS); bar: 50 μm; insert bar: 25 μm) and (b) yeast cells were quantified (yeast/mm² lung) using magnifications ×400. Fungal burden was quantified from tissue homogenates and expressed as the number of colony-forming units (CFU) per gram of tissue CFU/g in lung (c) and CFU per spleen (d). Data are representative of one of the two experiments performed independently (n = 10 per group). Values are mean ± SEM. ^# p < 0.05 WT versus Lgals1^−/−, both infected.

Figure 3

Lgals1^−/− mice have increased neutrophil infiltration in the lung parenchyma. H. capsulatum-infected mice were euthanized on day 15 after infection and lung sections (5 μm) were embedded in paraffin blocks. Lung sections from WT + H. capsulatum (I, bar: 100 μm; III, bar: 25 μm) and Lgals1^−/− + H. capsulatum (II, bar: 100 μm; IV, bar: 25 μm) were stained with hematoxylin (a) and neutrophils were quantified (neutrophils/mm²) using magnifications ×400 (b). Data are representative of one of the two experiments performed independently (n = 10 per group). Values are mean ± SEM. ^# p < 0.05 WT versus Lgals1^−/−, both infected.

Figure 4

Lgals1^−/− mice exhibit increased inflammatory response day 15 after infection with H. capsulatum. Cytokines IL-12p40 (a), TNF-α (b), IL-1-α (c), and NO₂ ⁻ (d) were quantified from homogenized lungs on day 15 after infection with H. capsulatum. Cytokines levels (pg/mL) were determined in the supernatants by ELISA and NO₂ ⁻ (μM) by using a Griess reaction. Data are representative of one of the two experiments performed independently (n = 10 per group). Values are mean ± SEM. ^∗ p < 0.05 infected mice versus control (uninfected), ^# p < 0.05 WT versus Lgals1^−/−, both infected.

Figure 5

Absence of endogenous Gal-1 increases prostaglandin PGE₂ production and Ptges2 expression in peritoneal macrophages. (a) In vivo prostaglandin E₂ was quantified in supernatants from homogenized lungs on day 15 after infection with H. capsulatum (5 × 10⁵ yeasts/mice) by ELISA. (b) 5 × 10⁵ peritoneal macrophages were incubated in vitro with H. capsulatum (MOI 1 : 1) during 2 and 24 hours and mRNA levels for Ptges2 were quantified and plotted as Fold Regulation by Log₂. In addition, PGE₂ was assessed in vitro in the supernatants by ELISA 24 hours after infection (c). In vivo data are representative of one of the two experiments performed independently (n = 10 per group). Values are mean ± SEM. ^∗ p < 0.05 infected mice versus control (uninfected), ^# p < 0.05 WT versus Lgals1^−/−, both infected.

Figure 6

Gal-1 does not bind and kill the yeast form of H. capsulatum. (a) Yeasts were incubated for 1 hour at 4°C with 1.0 μM and 4.0 μM biotinylated-hrGal-1, in the presence or absence of 20 mM lactose (Gal-1 inhibitor) or sucrose (control, noninhibitor). After that, yeasts were incubated with streptavidin-FITC and labeled cells were acquired on a FACS Canto (Becton Dickinson, Mountain View, CA, USA) and analyzed in the DIVA software (Becton Dickinson). (b) As a control, HL-60 cells (1 × 10⁶) were incubated with 1 μM biotinylated-hrGal-1 for 1 hour at 4°C, in presence or absence of 20 mM lactose or sucrose. (c) Several hrGal-1 concentrations (0.5, 1.0, 2.5, 4.0, and 10 μM) were incubated with 1 × 10⁶ H. capsulatum cells during 24 and 48 h. After each time, relative fluorescent units (RFU) (560–590 nm) were measured and represent yeast cells metabolically active through the dye resazurin reagent. Data are representative of two independent experiments and expressed as the mean ± SEM.