Therapeutic administration of KM+ lectin protects mice against Paracoccidioides brasiliensis infection via interleukin-12 production in a toll-like receptor 2-dependent mechanism

Abstract

KM(+) is a mannose-binding lectin from Artocarpus integrifolia that induces interleukin (IL)-12 production by macrophages and protective T helper 1 immune response against Leishmania major infection. In this study, we performed experiments to evaluate the therapeutic activity of jackfruit KM(+) (jfKM(+)) and its recombinant counterpart (rKM(+)) in experimental paracoccidioidomycosis. To this end, jfKM(+) or rKM(+) was administered to BALB/c mice 10 days after infection with Paracoccidiodes brasiliensis. Thirty days postinfection, lungs from the KM(+)-treated mice contained significantly fewer colony-forming units and little to no organized granulomas compared to the controls. In addition, lung homogenates from the KM(+)-treated mice presented higher levels of nitric oxide, IL-12, interferon-gamma, and tumor necrosis factor-alpha, whereas higher levels of IL-4 and IL-10 were detected in the control group. With mice deficient in IL-12, Toll-like receptor (TLR) 2, TLR4, or TLR adaptor molecule MyD88, we demonstrated that KM(+) led to protection against P. brasiliensis infection through IL-12 production, which was dependent on TLR2. These results demonstrated a beneficial effect of KM(+) on the severity of P. brasiliensis infection and may expand its potential use as a novel immunotherapeutic molecule.

Figure 1

Therapeutic effect of KM⁺ on P. brasiliensis infection. Mice inoculated with 10⁶ yeast cells were treated with jfKM⁺, rKM⁺, or PBS (as a control). The lung homogenates obtained from these mice were analyzed at 30 days postinfection. A: CFU. B–E: Cytokine levels. F: Nitrite concentrations. Assays were performed in triplicate and the results represent the mean ± SD of at least three independent experiments. *P < 0.05 versus control group.

Figure 2

Therapy with KM⁺ leads to resolution of the pulmonary lesions in P. brasiliensis-infected mice. Lung histopathology of P. brasiliensis-infected mice treated therapeutically with jfKM⁺ (B) or rKM⁺ (D). PBS was administered to control animals (A and C). The lung sections obtained on day 30 postinfection were H&E stained. The mice injected with PBS (A and C) presented extensive and confluent lesions, with epithelioid granulomas surrounding a large number of viable and nonviable yeast cells. The mice treated with jfKM⁺ (B) or rKM⁺ (D) presented no granulomas or yeast cells. Scale bars: A, B, D = 200 μm; C = 20 μm.

Figure 3

Leukocytes into the bronchoalveolar fluid of the P. brasiliensis-infected mice treated or not with KM⁺. A: Cells from BALF, obtained 30 days after infection, were categorized as neutrophils, lymphocytes, and macrophages according to morphological criteria. B: Lymphocytes from BALF were analyzed by flow cytometry after labeling with CD4, CD8, and CD19 cell markers. Total number of viable cells was determined by hemocytometer count and exclusion of trypan blue. The results represent the mean ± SD of five mice per group. *P < 0.05 versus control (PBS).

Figure 4

Therapeutic effect of KM⁺ on P. brasiliensis infection depends on IL-12 but not on NO. A–D: Lung sections from NOS2^−/− mice and their WT counterpart obtained at 14 days postinfection were H&E stained. The lung of NOS2^−/− or WT mice treated with jfKM⁺ (B and D) presented normal architecture and rare foci of mild inflammation, while the lung from mice given PBS presented confluent granulomas surrounding yeast cells (A and C). E–H: Lung sections from IL-12^−/− mice and their WT counterpart obtained at 30 days postinfection were H&E stained. The lung sections of IL-12^−/− mice treated (F) or not (E) with jfKM⁺ presented severe granulomatous inflammation reaction similar to those observed in WT mice injected with PBS (G). Infected WT mice treated with jfKM⁺ presented only very mild inflammatory reaction (H). Scale bars = 200 μm.

Figure 5

KM⁺ does not protect IL-12^−/− mice from P. brasiliensis infection but protects NOS2^−/− and WT mice evenly. NOS2^−/− (A) and IL-12^−/− (B and C) mice, as well as their WT counterpart, were intravenously infected with 10⁶ P. brasiliensis yeast cells and treated or not with jfKM⁺ 10 days postinfection. The number of viable yeasts (A and B) and the IFN-γ production (C) were determined by CFU and capture ELISA, respectively, on day 14 after infection for the NOS2^−/− mice and on day 30 for the IL-12^−/− mice. The results represent the mean ± SD of three mice per group and are representative of at least two experiments. *P < 0.05 versus control group (WT); **P < 0.05 versus WT control (PBS).

Figure 6

KM⁺ induces IL-12 production by macrophages in a TLR2-dependent mechanism. Peritoneal macrophages from TLR2^+/+ (C57BL/6), TLR2^−/−, C3H/HePas (TLR4^lps-n, expressing a functional TLR4), and C3H/HeJ (TLR4^lps-d, expressing a nonfunctional TLR4) mice were cultured for 48 hours in the presence or absence of jfKM⁺ (5 μg/ml), zymosan (10 μg/ml), or lipopolysaccharide (10 μg/ml). In some experiments, macrophages were cultured with jfKM⁺ in the presence of 50 mmol/L d-mannose (KM⁺/D-Man). The amount of IL-12p70 in the supernatant was determined by capture ELISA. Assays were performed in triplicate and the results represent the mean ± SD of at least three independent experiments. *P < 0.05 versus control group; #P < 0.05 versus positive control (KM⁺). ND, not detected.