The Capsular Polysaccharides GXM and GXMGal from Cryptococcus neoformans Modulate Macrophages Infected with Leishmania major

Abstract

Leishmania spp. are obligatory intracellular parasites that primarily infect macrophages. The macrophage immune response plays a pivotal role in determining the control or progression of infection. "M1-like" macrophages mediate parasite clearance through the production of nitric oxide, pro-inflammatory cytokines, and reactive oxygen species, whereas "M2-like" macrophages contribute to infection progression by exerting anti-inflammatory effects. The capsular polysaccharides Glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal) from Cryptococcus neoformans are capable of immunomodulating the macrophage response. GXM exhibits immunoregulatory activity, whereas GXMGal induces a pro-inflammatory response. Although the activity of these polysaccharides has been studied in cryptococcosis, their immunomodulatory potential in other infectious models remains largely unexplored. Here, we investigated the effects of GXM and GXMGal on Leishmania major infection in murine peritoneal macrophages. Murine peritoneal macrophages were infected with L. major and, 24 h post-infection, treated with 50 μg of either GXM or GXMGal. Our data revealed that GXM treatment enhanced L. major infection, while GXMGal treatment had no significant effect on the parasitic load in infected macrophages.

Keywords: GXM; GXMGal; Leishmania major; capsular polysaccharides; immunomodulatory; infection; macrophages.

Figure 1

Cell viability assay. Peritoneal macrophages were treated for 24 h with different concentrations of GXM or GXMGal. Serial dilutions were performed to obtain concentrations as low as 2.34 μg/mL, as shown in the figure. After 24 h of treatment, cell viability was assessed using MTT assay. Absorbance was measured with a microplate reader at 550 nm. All cultures were performed in triplicate, and the data are representative of three independent experiments. Bars represent the mean ± SD. Statistical analysis was performed using One-Way ANOVA.

Figure 2

Quantification of the parasitic load of promastigotes and amastigotes in murine macrophages infected with L. major. (a–c) Murine peritoneal macrophages (5 × 10⁵/mL) were infected with promastigote forms of L. major (5 × 10⁶/mL) in 48-well plates at a 10:1 parasite/macrophage ratio. After 24 h of infection, the cultures were treated with 50 μg/mL of GXM or GXMGal in the presence or absence of IFN-γ (20 ng/mL) and incubated for 3 days. (a,b) On the third day, the cells were stained with a panoptic staining kit to determine the percentage of infected macrophages (a) and the number of amastigotes/macrophages (b). (c) On the third day, the cells were transferred to Schneider’s supplemented medium, and the plate was maintained in a B.O.D. incubator at 27 °C in a humid chamber for 12 days. The number of viable promastigotes was quantified by counting in a Neubauer chamber. (a–c) All experiments were performed in triplicate, and the data were representative of 3 independent experiments. Data is presented as mean ± SD. Statistical analysis was conducted using One-Way ANOVA. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Figure 3

Measurement of cytokine production. (a–c) Peritoneal macrophages were infected with metacyclic promastigotes of L. major in 48-well plates at a multiplicity of infection (MOI) of 10:1. After 24 h of infection, cultures were treated with either GXM or GXMGal. Cytokine levels were quantified 24 h post-treatment using commercial ELISA kits, according to the manufacturer’s recommendation. Absorbance was measured at 450 nm. (d–f) Peritoneal macrophages infected with L. major and treated or not with GXM or GXMGa, were incubated in the presence or absence of neutralizing monoclonal antibodies targeting IL-10, TGF-β, or TNF-α, as well as an isotype control. After 3 days of incubation, cells were transferred to Schneider’s medium supplemented with 2% human urine and 10% FBS and subsequently maintained at 27 °C in a B.O.D. incubator within the humid chamber for 7 days. The number of viable promastigotes was determined using a Neubauer chamber. All cultures were performed in triplicate. Data were presented as mean ± SD. Statistical analysis was performed by One-Way Anova. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Figure 4

Measurement of nitrite. Peritoneal macrophages were infected with L. major promastigotes in 96-well plates at a multiplicity of infection (MOI) of 10:1. After 24 h of infection, cultures were treated with GXM or GXMGal in the presence or absence of IFN-γ. Nitrite production was assessed 6 h after treatment using the Griess method (a). The production of NO was inhibited using the inhibitor L-NIL (1 mM) (b). All conditions were performed in triplicate, and the data are presented as mean ± SD. Statistical analysis was performed by One-Way Anova. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Figure 5

Measurement of PGE₂ in peritoneal macrophages infected with L. major promastigotes. Murine peritoneal macrophages (2 × 10⁵/mL) were infected or not with L. major promastigote (2 × 10⁶/mL; MOI 10:1) in 48-well plates. After 24 h of infection, the cultures were treated with 50 µg/mL of GXM or GXMGal. PGE₂ levels were measured 24 h post-treatment using a commercial ELISA kit, according to the manufacturer’s instructions. All cultures were performed in triplicate, and the data are representative of three independent experiments. Data represents the mean ± SD. Statistical analysis was conducted using One-Way ANOVA. *** p ≤ 0.001, ** p ≤ 0.01.

Figure 6

Inhibition of PGE₂ synthesis modulates parasite burden in macrophages treated with GXM or GXMGal. Peritoneal macrophages, infected or not with L. major, were treated with (a) aspirin (10 mg/mL) or (b) NS-398 (1 mM) in the presence of GXM or GXMGal. After 24 h of treatment, cells were transferred to Schneider’s medium supplemented with 2% human urine and 10% FBS, and the plate was kept in a B.O.D. incubator at 27 °C in a humid chamber for 7 days. Promastigotes were quantified using a Neubauer chamber. All cultures were performed in triplicate, and bars represent mean ± SD. Statistical analysis was conducted by One-Way ANOVA. *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 7

Lipid droplet quantification. Peritoneal macrophages were infected with L. major promastigotes (MOI 10:1). After 24 h of infection, cells were treated or not with GXM or GXMGal on coverslip. Following 24 h of treatment, cells were fixed with 3.7% paraformaldehyde for 30 min and subsequently stained with osmium tetroxide. Lipid droplets were quantified in 100 cells using optical microscope. All cultures were performed in triplicate, and the bars represent mean ± SD. Statistical analysis was performed by One-Way Anova. ** p ≤ 0.01.