Evidence for Asthma in the Lungs of Mice Inoculated with Different Doses of Toxocara canis

Abstract

Toxocara canis, a common roundworm that mainly causes toxocariasis, is a zoonotic parasite found worldwide. Humans, an accidental host, can acquire T. canis infection through accidental ingestion of T. canis-embryonated egg-contaminated food, water, and soil, and by encapsulated larvae in a paratenic host's viscera or meat. Long-term residence of T. canis larvae in a paratenic host's lungs may induce pulmonary inflammation that contributes to lung injury, airway inflammatory hyperresponsiveness, and collagen deposition in mice and clinical patients. This study intended to investigate the relationship between T. canis infection and allergic asthma in BALB/c mice inoculated with high, moderate, and low doses of T. canis eggs for a 13-week investigation. The airway hyperresponsiveness (AHR) to methacholine, collagen deposition, cytokine levels, and pathological changes in lung tissues was assessed in infected mice at weeks 1, 5, and 13 postinfection. The cell composition in bronchoalveolar lavage fluid of infected mice was assessed at weeks 5 and 13 postinfection. Compared with uninfected control mice, all groups of T. canis-infected mice exhibited significant AHR, a dose-dependent increase in eosinophilic infiltration leading to multifocal interstitial and alveolar inflammation with abundant mucus secretion, and collagen deposition in which the lesion size increased with the infective dose. Infected mice groups also showed significant expressions of eotaxin and type 2 T-helper-dominant cytokines such as interleukin (IL)-4, IL-5, and IL-13. Overall, these results suggest that T. canis larval invasion of the lungs may potentially cause pulmonary inflammatory injury and could subsequently contribute to the development of allergic manifestations such as asthma.

Figure 1.

Toxocara canis infection of mice induced the development of airway hyperresponsiveness and abundant infiltrating cells. (A) Airway resistance was measured in response to increasing concentrations of methacholine (0–32 mg/mL) by invasive-body plethysmography. (B) Cellular composition of the bronchoalveolar lavage fluid (BALF) of mice. After measuring pulmonary function parameters, each group of mice was sacrificed, and cells in the BALF were collected, counted, and classified as Eos = eosinophils; Lym = lymphocytes; Macro = macrophages; and Neu = neutrophils. Results are expressed as the mean ± standard error of the mean (n = 8 mice in the control group and n = 8 mice in all three experimental groups) of pulmonary resistance (R_L) after methacholine challenge and cell numbers in the BALF. * P < 0.05, ** P < 0.01, *** P < 0.001, and ***** P < 0.0001 with a one-way analysis of variance for pulmonary resistance (R_L) and (t-test) vs. the control group for cell populations. This figure appears in color at www.ajtmh.org.

Figure 2.

Toxocara canis infection increased inflammatory cell infiltration around the airway wall at 1, 5, and 13 weeks postinfection, and the thickness of the layers, destruction of cell networks, and inflammatory cell composition (including neutrophils, macrophages, eosinophils, and lymphocytes) which induced airway narrowing were evaluated. Sections were stained with hematoxylin and eosin for the morphological analysis. Tissues were examined by light microscopy (scale bars: ×100 magnification, 100 μm; ×400 magnification, 50 μm). This figure appears in color at www.ajtmh.org.

Figure 3.

Toxocara canis infection enhanced mucus production in the lungs. (A) Sections were stained with periodic acid–Schiff (PAS) stain for the morphological analysis. Epithelial cells were replaced by blue/purple-stained goblet cells. Tissues were examined by light microscopy (scale bars: ×100 magnification, 100 μm; ×400 magnification, 50 μm). (B) Quantification of PAS staining. Results are expressed as the mean ± standard error of the mean (n = 8 mice in the control group and n = 8 mice in all three experimental groups) of the percentage of the area occupied by mucus production. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (t-test) vs. the control group. This figure appears in color at www.ajtmh.org.

Figure 4.

Toxocara canis infection caused collagen deposition in mouse lung tissues. (A) Sections were stained with Masson’s trichrome (MT) stain for the morphological analysis. In MT staining, blue staining of subepithelial collagen was observed. Tissues were examined by light microscopy (scale bars: ×100 magnification, 100 μm; ×400 magnification, 50 μm). (B) Quantification of MT staining. Results are expressed as the mean ± standard error of the mean (n = 8 mice in the control group and n = 8 mice in all three experimental groups) of the percentage collagen deposition. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (t-test) vs. the control group. This figure appears in color at www.ajtmh.org.

Figure 5.

Type 2 T-helper-type cytokine expressions, including interleukin (IL)-4, IL-5, IL-13, and eotaxin, increased in Toxocara canis–infected mice inoculated with low-, moderate-, and high-dose infections at 1, 5, and 13 weeks postinfection. (A) Type 2 T-helper-type cytokine accumulation in lung tissues was analyzed by Western blotting. (B) Quantification of cytokine accumulation. Results are expressed as the mean ± standard error of the mean (n = 8 mice in the control group and n = 8 mice in all three experimental groups) of relative multiples of the control. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (t-test) vs. the control group. NS = no significant difference.