Fig 1.
Kinetics of immune response and parasite burden during experimental ascariasis in mice.
Number of leukocytes and their cell subpopulations in bronchoalveolar lavage fluid (BAL) (n = 7) (A). Coincidence curve showing inverse relationship observed between total parasite burden and the number of eosinophils (n = 7) (B) and total SIgA levels (n = 7) (C) in BAL. Scatterplot graphics showing a positive correlation between total SIgA levels and the number of eosinophils from BAL (n = 7) (D) and a negative correlation between total parasite burden and total SIgA levels in BAL (n = 7) (E) on the8th dpi. Schematic representation of experimental design (F). One-way ANOVA followed by Tukey´s multiple comparisons test was used to evaluate differences between groups in (A), and Pearson’s Linear Regression was used to evaluate the correlation in (D) and (E). The letters in (B) and (C) indicate statistically significant difference between mean values, where [a < b < c < d] and [a’ < b’ < c’]. Significant differences between groups (p < 0.05) are represented by the p values in A, D, and E graphs. Data are presented as mean ± SEM.
Fig 2.
TLR2 and TLR4 receptors trigger eosinophilia and mucosal SIgA production that control larval ascariasis.
Schematic representation of the experimental design using TLR2-/-, TLR4-/- and WT C57BL/6 mice (A). Number of eosinophils recovered from BAL (n = 14) (B), total SIgA levels from BAL (n = 14) (C) and intestine lavage (n = 14) (D) on the 8th dpi. Total parasite burden, that is, the number of larvae recovered in lung parenchyma and airways of WT C57BL/6, TLR2-/- and TLR4-/- mice on the 8th dpi (n = 14) (E). Effect of probiotic Lactococcus lactis (NCDO 2118) on immunological and parasitological parameters during the acute phase of experimental ascariasis in mice, namely, number of eosinophils in BAL (n = 7), total SIgA (n = 7), and coincident reduction in parasite burden (n = 7) (F). Two-way ANOVA followed by Sidak´s multiple comparisons test was used to evaluate differences between groups (B-E) and the Mann-Whitney test was used to assess differences between the groups (F). Differences were considered significant at p ≤ 0.05 and are represented by symbols in the graphs. * represents differences between knockout and WT C57BL/6 mice groups and # represents differences between non-infected and single-infected groups from same strain (B, C, and D); and, the * represents differences between the single-infected and non-infected group and & represents differences between the NCDO and M17 groups (F). [1 symbol = p < 0.05], [2 symbols = p < 0.01], [3 symbols = p < 0.001]. Data are presented as mean ± SEM.
Fig 3.
Eosinophils are required for SIgA production and parasite burden control in mice.
Schematic representation of the experimental design of a single-infection model using WT BALB/c and GATA1-/- mice (eosinophil deficient) (n = 5) (A). Number of larvae recovered from the liver, lung, and intestine (B) of WT BALB/c and GATA1-/- mice on the 4th, 8th, 10th, and 12th dpi. Levels of total and A. suum antigen-specific SIgA from BAL on the 8th dpi (C). T-test was used to assess differences between the groups in (B); one-way ANOVA followed by Tukey´s multiple comparisons test was used to evaluate differences between groups in (C). Differences were considered significant at p ≤ 0.05 and are represented by symbols in the graphs. * represents differences between the single- and non-infected group’s mice of the same strains, and & represents differences between the WT and GATA1-/- mice with the same treatment [1 symbol = p < 0.05], [2 symbols = p < 0.01]. Data are presented as mean ± SEM.
Fig 4.
Eosinophils are important in probiotic modulation after infection.
Schematic representation of single-infection model using WT BALB/c and GATA1-/- mice after treatment with Lactococcus lactis NCDO (2118) (n = 6) (A). Total SIgA levels in BAL on the 22nd day after treatment with probiotic and 8 days after infection (n = 6) (B). Number of larvae recovered from WT BALB/c and GATA1-/- mice lungs (C). Differences between groups were statistically evaluated by the following tests: Two-Way ANOVA followed by Sidak´s multiple comparisons test was used to evaluate differences between groups in (B); One-Way ANOVA followed by Tukey´s multiple comparisons test was used to evaluate differences between groups in (C). Differences were considered significant at p ≤ 0.05 and are represented by symbols in the graph (C) where the * = p < 0.05. Data are presented as mean ± SEM.
Fig 5.
Eosinophils contribute to pulmonary inflammation during A. suum single-infection in mice.
Histopathological analysis of the lesion caused by larval ascariasis and lung inflammation in WT BALB/c and GATA1-/- mice on the 8th and 12th dpi (n = 6). Representative microphotography of lung tissue stained with H&E (Bar scale = 50 μm) (A-D). Number of total leukocytes recovered from BAL on the 8th dpi (n = 6) (E). Number of eosinophils in situ in the lung at 0, 4, 8 and 12 dpi (n = 5) (F). Optical density representing Eosinophil peroxidase (EPO) and neutrophil myeloperoxidase (MPO) activity in the lung at 0, 4, 8, and 12 dpi (n = 12) (G and H). Levels of cytokines in the lung at the 12th dpi (n = 6) (I—Q). Data are represented as mean ± SEM. One-way ANOVA followed by Tukey´s multiple comparisons test was used to evaluate differences between groups (G, I, J, K, L, N, O, and P). Kruskal-Wallis test followed by Dunn´s multiple comparisons test was used to evaluate differences between groups (H, M, and Q). The p values are represented by symbols in the graphs wherein * represents differences between the non-infected group, * represents differences between same groups in the 8 dpi, # represents differences between groups from different strains that received the same treatment. [1 symbol = p < 0.05], [2 symbols = p < 0.01], [3 symbols = p < 0.001] and [4 symbols = p < .0001].
Fig 6.
Pulmonary mechanical dysfunction and fibrosis induced by larval ascariasis in the lungs.
Representative photomicrographs of single-infected WT BALB/c and GATA1-/- mice (n = 5): Fibrosis analysis using Masson’s trichrome technique (stains collagen fibers blue) and Picrosirius Red (stains collagen fibers red) performed at the 8th dpi (A, J and B, K) showing normal histological appearance with few collagen fibers and at 12th dpi (C, L, and D, M) showing increase in collagen deposition in single-infected WT BALB/c and GATA1-/- mice. Histological analysis at the 12th dpi using Picrosirius Red with polarized light showing increased deposition of type I collagen fibers (stained red, P and Q), distinct from type III collagen observed at the 8th dpi (stained green and yellow, N and O). Assessment of invasive pulmonary spirometry of Ascaris single-infected WT BALB/c and GATA1-/- mice through the following parameters: Forced Vital Capacity, FVC (E), Lung Resistance, Rl (F), Dynamic Compliance, Cdyn (G), Inspiratory Capacity, IC (H), Forced Expiratory Volume at 100 msec, FEV100 (I). Data are represented as mean ± SEM. Significant differences between GATA1-/- and WT BALB/c groups at the 12th dpi are represented in the graph by p values. P values are represented by symbols in the graphs wherein * represents differences between non-infected groups from the same strain, * represents differences between the single-infected groups from the same strain, # represents differences between groups from different strains that received the same treatment. One-way ANOVA followed by Tukey´s multiple comparisons test was used to evaluate differences between groups. [1 symbol = p < 0.05], [2 symbols = p < 0.01], [3 symbols = p < 0.001] and [4 symbols = p < 0.0001]. Bar scale = 50 μm.
Fig 7.
Eosinophils contribute to parasite burden control and SIgA production during larval ascariasis after multiple exposures.
Experimental design of A. suum multiple infections (A). Number of larvae in WT BALB/c and GATA1-/- mice lung parenchyma and bronchoalveolar lavage (BAL) (n = 14) (B). Number of total leukocytes and their subpopulations, namely, lymphocytes, macrophages, neutrophils, eosinophils, and total protein and hemoglobin levels in BAL (n = 6) (C). All immunological and parasitological parameters were evaluated at the 8th dpi. Data are represented as mean ± SEM. Significant differences (p < 0.05) between are represented in the graph by p symbols. * represents differences between non-infected groups from the same strain, * represents differences between the single-infected groups from the same strain, # represents differences between groups from different strains that received the same treatment. [1 symbol = p < 0.05], [2 symbols = p < 0.01], [3 symbols = p < 0.001] and [4 symbols = p < .0001].
Fig 8.
Effect of eosinophil deficiency in IgA production in lungs after larval ascariasis.
Total SIgA and Ascaris-specifics SIgA levels in bronchoalveolar lavage (BAL) (n = 5) (A). Representative microphotography of IgA immunohistochemistry in lung tissues (B-G) showing immunostained cells predominantly in the peribronchial spaces and the bronchial epithelium. Non-infected WT BALB/c (B), non-infected GATA1-/- (C), single-infected WT BALB/c (D), single infected GATA1-/- (E), re-infected WT BALB/c (F), and re-infected GATA1-/- (G) (n = 6). IgA is marked in brown. Arrows indicate positive markings on the bronchial epithelium. Bar scale = 40 μm).
Fig 9.
Effect of eosinophil deficiency on chronic pulmonary inflammation induced by multiple A. suum infections.
Representative microphotography of lungs tissues stained with H&E and observed on the 8th dpi at low magnification (Bar scale = 100 μm) (A-D) and at high magnification (Bar scale = 50 μm) (E-H), showing histopathological lesions in the single-infected group of WT BALB/c (A and E) and GATA-/- (B and F) (n = 6), and re-infected WT BALB/c (C and G) and GATA1-/- groups (D and H) (n = 6). Thickened septa are indicated with arrowheads, inflammatory infiltrates are indicated with ‘#’, hemorrhage is indicated with ‘*’. Optical density represents eosinophil peroxidase (EPO) and neutrophil myeloperoxidase (MPO) activity in the lung tissue at the 8th dpi (n = 10) (I). Tissue cytokine profile in the 8th dpi (n = 10) (J). Differences between groups were statistically evaluated using one-way ANOVA followed by Tukey´s multiple comparisons test for the data represented in (I) and the Kruskal-Wallis followed by Dunn’s multiple comparisons test in the data represented in (J). Data are represented as mean ± SEM. P values are represented by symbols in the graphs wherein * represents differences between non-infected groups, * represents differences between single-infected groups, # represents differences between groups from different strains that received the same treatment. [1 symbol = p < 0.05], [2 symbols = 0.01 > p > 0.05], [3 symbols = 0.001 > p > 0.01] and [4 symbols = p < 0.0001].
Fig 10.
Effect of eosinophil deficiency in lung injury, fibrosis, and dysfunction induced by multiple larval ascariasis.
Representative microphotography of fibroplasia/fibrosis histopathological analysis from lung tissues stained with Gomori’s trichrome (A-D) showing lesions and inflammation caused by larval ascariasis in single-infected (A-B) compared to re-infected (C-D) WT BALB/c (A and C) and GATA1-/- (B and D) mice (n = 6). Fibrosis is marked in bluish green. Bar scale = 50 μm. The area of fibrosis is represented by ‘*’. Bar graphs from morphometric analysis of the area of the lesion (E) and fibrosis (F) caused by larval migration and lung inflammation (n = 6). Assessment of lung mechanics in mice after single and multiple infections with A. suum (G) (n = 6). Data are represented as mean ± SEM. One-way ANOVA followed by Tukey´s multiple comparisons test was used to evaluate differences between groups in (E) and (F). Kruskal-Wallis test followed by Dunn´s multiple comparisons test was used to evaluate differences among groups in (G). Statistical differences are represented by symbols in the graphs, where * represents differences observed between non-infected groups, * represents differences between single-infected groups, # represents differences between groups of different strains that received the same treatment. [1 symbol = p < 0.05], [2 symbols = 0.01 > p > 0.05], [3 symbols = 0.001 > p > 0.01] and [4 symbols = p < 0.0001]. All experiments were analyzed at the 8th dpi.