Fig 1.
Repeat Ascaris challenge by oral gavage reduces Ascaris worm intensity in the liver with associated impaired host gastric microenvironment function.
Repeat Ascaris infection mouse model (A) in which wild-type mice were infected with embryonated Ascaris eggs or PBS twice a week for two weeks by oral gavage (o.g.). Four days following the last infection mice were euthanized and stomach and liver tissue were harvested. Larval count in the liver (B) illustrating decreased intensity in the repeat Ascaris infection mouse model compared to a single Ascaris infection model. mRNA relative gene expression (C) of chia 1 (left) and atp4a (right) normalized to 18s in the stomach by qPCR following repeated Ascaris infection compared to PBS, non-infected controls. Western blot with quantification (D) of protein expression of Atp4a and AMCase in the stomach following repeated Ascaris infection compared to PBS, non-infected controls. Relative expressions of both AMCase and Atp4a were calculated based on densitometry normalization to the first control sample. (n ≥ 4, mean±S.E.M, *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed Student’s t-test. Data are shown as representative of two independent experiments. Illustration created by biorender.com.).
Fig 2.
Reduced worm intensity is independent of the gastric adaptive immune response following repeated Ascaris challenge.
Repeat Ascaris infection mouse model in which wild-type mice were infected with embryonated Ascaris eggs or PBS twice a week for two weeks by oral gavage. Four days following the last infection, mice were euthanized and stomach tissue was harvested. Repeat Ascaris infection is associated with reduced expression of T cell recruitment and activation genes in gastric tissue relative to infection-naïve mice represented by Z-scores (A) from bulk RNA sequencing. mRNA relative expression (B) of T cell recruitment and activation genes from A in gastric tissue by qPCR normalized to the first control sample. Flow cytometry with quantification (C) showing concentration of CD4+ T cells in gastric tissue. Repeat Ascaris infection mouse model (D) in which mice deficient in T and B cells (RAG2-/-) were infected with embryonated Ascaris eggs or PBS twice a week for two weeks by oral gavage. Four days following the last infection, mice were euthanized and liver tissue was harvested. Larval count in the liver of RAG2-/- mice (E) illustrating decreased infection intensity for repeat Ascaris infection mouse model compared to a single Ascaris infection despite loss of adaptive immunity. (n ≥ 3, mean±S.E.M, *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed Student’s t-test. Data are shown as representative of two independent experiments. Illustration created by biorender.com.).
Fig 3.
Repeated Ascaris challenge causes cellular changes to the gastric mucosa.
Bulk RNA sequencing shows differential expression represented by z-score (A) for genes in stomach tissue associated with cellular reprogramming and development of pyloric metaplasia following mucosal injury including relatively decreased expression of atp4a and atp4b and increased expression of tff2 in the repeat Ascaris mouse models compared to naïve controls. Bulk RNA sequencing in stomach tissue also reveal (B) increased expression (represented by Z-score) of genes involved in cellular apoptosis pathways in repeat Ascaris infected mice compared to uninfected controls. Immunohistochemistry (C) shows increased apoptotic bodies (insert) consistent with parietal cell death identified by histopathology (H&E staining) in wild-type repeat Ascaris challenge model compared to wild-type naive mice (Scale bar: 100μm for 100x, 25 μm for insert). Quantification of apoptotic bodies (D) in the gastric mucosa supports increased cell death in the gastric mucosa following repeat Ascaris challenge compared to naive mice. Immunohistochemistry (E) demonstrates increased expression of tff2 (brown staining, Scale bar: 100μm) and qPCR (F) quantification of increased tff2 mRNA relative expression normalized to 18s in the gastric mucosa in repeated Ascaris challenge model compared to naive mice. (n = 4, mean±S.E.M, **p < 0.01, using two-tailed Student’s t-test. Magnification: 100× and 400× with 5 × zoom in. Scale bar: 100μm and 25 μm. Data are shown as representative of two independent experiments.).