Novel gene expression responses in the ovine abomasal mucosa to infection with the gastric nematode Teladorsagia circumcincta

Abstract

Infection of sheep with the gastric nematode Teladorsagia circumcincta results in distinct Th2-type changes in the mucosa, including mucous neck cell and mast cell hyperplasia, eosinophilia, recruitment of IgA/IgE producing cells and neutrophils, altered T-cell subsets and mucosal hypertrophy. To address the protective mechanisms generated in animals on previous exposure to this parasite, gene expression profiling was carried out using samples of abomasal mucosa collected pre- and post- challenge from animals of differing immune status, using an experimental model of T. circumcincta infection. Recently developed ovine cDNA arrays were used to compare the abomasal responses of sheep immunised by trickle infection with worm-naïve sheep, following a single oral challenge of 50 000 T. circumcincta L3. Key changes were validated using qRT-PCR techniques. Immune animals demonstrated highly significant increases in levels of transcripts normally associated with cytotoxicity such as granulysin and granzymes A, B and H, as well as mucous-cell derived transcripts, predominantly calcium-activated chloride channel 1 (CLCA1). Challenge infection also induced up-regulation of transcripts potentially involved in initiating or modulating the immune response, such as heat shock proteins, complement factors and the chemokine CCL2. In contrast, there was marked infection-associated down-regulation of gene expression of members of the gastric lysozyme family. The changes in gene expression levels described here may reflect roles in direct anti-parasitic effects, immuno-modulation or tissue repair.

Figure 1

Venn diagrams to illustrate total numbers of genes whose expression levels were significantly altered (FDR ≤ 0.05) detected in each hybridisation experiment; 1a) Expt.1; day 5 post-challenge 1b) Expt. 2; day 2 post-challenge. Nv = "naïve" yearlings, worm naïve prior to challenge; Im = "immune" yearlings, previously infected by trickle infection prior to challenge; d0, d2, d5 = days post-challenge; (details in Table 1). The figures in the circled regions correspond to the number of genes with significantly altered (FDR ≤ 0.05) expression levels in each comparison (details given in Table 2) while the figures in the overlapping regions correspond to the number of genes whose expression levels were significantly altered (FDR ≤ 0.05) in two or more comparisons; eg. 8 genes showed significantly altered expression levels in both the Imd2/Imd0 and Imd0/Nvd0 comparisons. The figures outside the circled regions correspond to the numbers of genes on the array with no significant change in expression level in any of the comparisons.

Figure 2

The top 20 most significant biological functions identified from the Imd0/Nvd0 and Imd2/Imd0 datasets from Expt. 2, using Ingenuity Pathways Analysis software (Ingenuity^® Systems, [26]). The Functional Analysis identified the biological functions and/or diseases that were most significant to the data set. Right-tailed Fisher's exact test was used to calculate a P-value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone.

Figure 3

Key changes detected in Expt. 1 (day 5 post-challenge) and Expt. 2 (day 2 post-challenge). Summary of mean infection-associated fold changes in the expression levels of key groups of transcripts detected from the microarray analyses. Transcripts are grouped as follows according to associated function/cell type; a) cytotoxicity, b) mucus composition, c) heat shock response, d) proinflammatory response, e) tissue remodelling (matrix metalloproteinases and inhibitors), f) nutritional (lysozyme family), g) unknown (MALAT). The groups being compared are indicated on the y-axis.

Figure 4

Results of competitive multiplex qRT-PCR analysis to investigate temporal changes in the expression levels of transcripts normally associated with cells exhibiting cytotoxicity; granulysin, granzymes A, B and H, and cathepsin C, as indicated, throughout the experiment trials summarised in Table 1. Open circles represent data from naïve sheep; closed triangles represent data from immune ("previously infected") sheep. Significant difference between Nv and Im at same timepoint; *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.005; Significant difference between timepoint and day 0 of same group (where available); ⁺P ≤ 0.05, ⁺⁺P ≤ 0.005, ⁺⁺⁺P ≤ 0.005 (Mann-Whitney U-test for non-parametric data). NB.- data for granzyme H- model fitting failed in some samples, so n = 5 in some groups as indicated.

Figure 5

Results of competitive multiplex qRT-PCR analysis to investigate temporal changes in the expression levels of mucous-cell associated transcripts; CLCA1 and ITLNs 1-3, as indicated, throughout the experiment trials summarised in Table 1. Open circles represent data from naïve sheep; closed triangles represent data from immune ("previously infected") sheep. Significant difference between Nv and Im at same timepoint; *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.005; Significant difference between timepoint and day 0 of same group (where available); ⁺P ≤ 0.05, ⁺⁺P ≤ 0.005, ⁺⁺⁺P ≤ 0.005 (Mann-Whitney U-test for non-parametric data).

Figure 6

Results of competitive multiplex qRT-PCR analysis to investigate temporal changes in the expression levels of a number of transcripts associated with tissue remodelling or inflammatory responses throughout the experiment trials summarised in Table 1. Data is shown for heat shock proteins HSPA8, HSPCA and STIP1; transcripts associated with pro-inflammatory responses CCL2, PLA2G2A and CF1, and matrix metalloproteinases and inhibitors MMP-13, MMP-23 and cystatin C, as indicated. Open circles represent data from naïve sheep; closed triangles represent data from immune ("previously infected") sheep. Significant difference between Nv and Im at same timepoint; *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.005; Significant difference between timepoint and day 0 of same group (where available); ⁺P ≤ 0.05, ⁺⁺P ≤ 0.005, ⁺⁺⁺P ≤ 0.005 (Mann-Whitney U-test for non-parametric data).

Figure 7

Results of competitive multiplex qRT-PCR analysis to investigate temporal changes in the expression levels of transcripts for lysozymes 1A and 4A, and MALAT, as indicated, throughout the experiment trials summarised in Table 1. Open circles represent data from naïve sheep; closed triangles represent data from immune ("previously infected") sheep. Significant difference between Nv and Im at same timepoint; *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.005; Significant difference between timepoint and day 0 of same group (where available); ⁺P ≤ 0.05, ⁺⁺P ≤ 0.005, ⁺⁺⁺P ≤ 0.005 (Mann-Whitney U-test for non-parametric data).