Th2-biased immune responses to body migrating Ascaris larvae in primary infection are associated with pathology but not protection

Abstract

Helminth infections lead to an overdispersion of the parasites in humans as well as in animals. We asked whether early immune responses against migrating Ascaris larvae are responsible for the unequal distribution of worms in natural host populations and thus investigated a susceptible versus a resistant mouse strain. In mice, the roundworm larvae develop until the lung stage and thus early anti-Ascaris immune responses against the migrating larvae in the liver and lung can be deciphered. Our data show that susceptible C57BL/6 mice respond to Ascaris larval migration significantly stronger compared to resistant CBA mice and the anti-parasite reactivity is associated with pathology. Increased eosinophil recruitment was detected in the liver and lungs, but also in the spleen and peritoneal cavity of susceptible mice on day 8 post infection compared to resistant mice. In serum, eosinophil peroxidase levels were significantly higher only in the susceptible mice, indicating functional activity of the recruited eosinophils. This effect was associated with an increased IL-5/IL-13 production by innate lymphoid cells and CD4⁺ T cells and a pronounced type 2 macrophage polarization in the lungs of susceptible mice. Furthermore, a comparison of wildtype BALB/c and eosinophil-deficient dblGATA-1 BALB/c mice showed that eosinophils were not essential for the early control of migrating Ascaris larvae. In conclusion, in primary infection, a strong local and systemic type 2 immune response during hepato-tracheal helminth larval migration is associated with pathology rather than protection.

Figure 1

Increased susceptibility to A. suum larval migration leads to enhanced hepatic and pulmonary inflammation. (a) Ascaris suum life stages in the mouse model. (b, c) Larval loads in liver and bronchoalveolar lavage (BAL). (d, e) Hematoxylin and eosin stained liver and lung sections at day 4 and 8 p.i. Arrows indicate the cellular infiltration. Bar graphs compile 3 independent experiments. (f) Percentages of the infective Ascaris egg inoculum released with feces within 6 h p.i. Data shown are pooled from 3 independent experiments with n = 3 animals per time point. p < 0.05 *; p < 0.01 **; p < 0.001 ***; p < 0.0001 ****.

Figure 2

Susceptible C57BL/6 mice presents local and systemic eosinophilic infiltration and degranulation during larval migration. (a) Exemplary gating strategy used to identify eosinophils (Gr-1^-Siglec-F⁺MHCII^-) in liver tissue and frequencies of hepatic eosinophils. (b) Liver sections, eosinophils depicted with black arrows and numbers of eosinophils in the liver. (c) Exemplary gating strategy used to identify eosinophils (Gr-1^-CD11b⁺Siglec F⁺) in lung tissue and frequencies of eosinophils in pulmonary CD45⁺ cells. (d) Lung sections and numbers of eosinophils in the lungs. (e, f) Frequencies of spleen eosinophils and peritoneal cavity eosinophils. (g) Eosinophil Peroxidase (EPO) levels detected in serum. Each symbol in bar plots represents an individual mouse. Data are pooled from 3 independent experiments with n = 3 to 6 animals per time point. p < 0.05 *; p < 0.01 **; p < 0.001 ***; p < 0.0001 ****.

Figure 3

Susceptible C57BL/6 mice present local activation of type 2 response and pulmonary M2 polarization during larval migration. (a, b) Frequencies of lung IL-5⁺/IL-13⁺ innate lymphoid cells (ILC) and CD4⁺ T cells. The percentage of cytokine positive ILC and CD4⁺ T cells is reported by red numbers. Black numbers report the frequencies of cytokine producing cells in live lymphocytes. (c) Exemplary gating of arginase 1 (Arg-1) and CD206 expression by CD11b⁺F4/80⁺Siglec-F^- monocyte-derived macrophages from the lung tissue. Bar graph reports the frequencies of lung monocyte-derived M2 macrophages in pulmonary CD45⁺ live cells. (d) Superimposed bar graphs depicting the frequencies of Arg-1⁺CD206⁺ alveolar macrophages (red bar graphs and grey dots) within the total population of lung alveolar macrophages (white dots). (e) Lung sections depicting the merged stains with DAPI (white), F4/80 (blue) and Arg-1 (red) and number of arginase 1 positive cells (Arg-1⁺) per 5 high power fields (HPF) in lung tissue. Data shown are pooled from 2 to 3 independent experiments with n = 3 to 6 animals per time point. p < 0.05 *; p < 0.01 **; p < 0.001 ***.

Figure 4

Lack of eosinophils does not alter the course of A. suum primary infection. (a) Larval load in bronchoalveolar lavage (BAL) (b) Frequencies of eosinophils in lung cells determined by flow cytometry. (c) Number of arginase 1 positive cells (Arg-1⁺ cells) in lung. (d) Frequencies of lung IL-5⁺/IL-13⁺ innate lymphoid cells (ILCs) and CD4⁺ T cells. (e) Liver and (f) lung histopathology scoring. Pooled data from 2 independent experiments with n = 3 to 5 animals per time point. p < 0.01 **. (g) Graphic summarizing the responses to body-migrating A. suum larvae in liver and lung in the susceptible and resistant mouse strain.