Identification of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite

Abstract

Infection of resistant or susceptible mice with Trichuris muris provokes mesenteric lymph node responses which are polarized towards Th2 or Th1, respectively. These responses are well documented in the literature. In contrast, little is known about the local responses occurring within the infected intestine. Through microarray analyses, we demonstrate that the gene expression profile of infected gut tissue differs according to whether the parasite is expelled or not. Genes differentially regulated postinfection in resistant BALB/c mice include several antimicrobial genes, in particular, intelectin (Itln). In contrast, analyses in AKR mice which ultimately progress to chronic infection provide evidence for a Th1-dominated mucosa with up-regulated expression of genes regulated by gamma interferon. Increases in the expression of genes associated with tryptophan metabolism were also apparent with the coinduction of tryptophanyl tRNA synthetase (Wars) and indoleamine-2,3-dioxygenase (Indo). With the emerging literature on the role of these gene products in the suppression of T-cell responses in vitro and in vivo, their up-regulated expression here may suggest a role for tryptophan metabolism in the parasite survival strategy.

FIG. 1.

Transcriptome analyses of Trichuris-infected gut tissue from resistant and susceptible mice at day 19 postinfection. The scatter plot shows the average log₂-fold changes in gene expression in gut tissue from AKR mice at day 19 compared to that from uninfected mice (x axis) versus the average log₂-fold changes in gene expression in gut tissue from BALB/c mice at day 19 compared to that from uninfected mice (y axis). Genes up-regulated in AKR and/or BALB/c gut tissue are labeled by gene symbol: indoleamine-2,3-dioxygenase (Indo), pancreatitis-associated protein (Pap), Ia-associated invariant chain (Ii), CXCL11 (Cxcl11), tryptophan tRNA synthase (Wars), T-cell-specific GTPase (Tgtp), Z-DNA binding protein 1 (Zbp1), CXCL9 (Cxcl9), coagulation factor VIII (F8), keratin-associated protein 5-4 (Krtap5-4), interferon alpha-inducible protein (G1p2), mouse mast cell protease 1 (Mcpt1), angiogenin like (Angl), intelectin (Itln), pancreatic lipase-related protein 2 (Pnliprp2), chloride channel calcium-activated 3 (Clca3), angiogenin-related protein (Angrp), and somatostatin (Sst). Genes regulated by an average log₂-fold change of ≥+0.9 or ≤−0.9 have a false discovery rate at this threshold of 2.9% by SAM analysis (44). Gut tissue was pooled from five individual animals within a group, and two independent infection experiments were run. The average log₂-fold changes presented represent values calculated from both infection experiments.

FIG. 2.

Altered levels of gene expression are still evident in chronically infected mice but not resistant mice at day 60 postinfection. The scatter plot shows the average log₂-fold changes in gene expression in gut tissue from AKR mice at day 60 compared to that from uninfected mice (x axis) versus the average log₂-fold changes in gene expression in gut tissue from BALB/c mice at day 60 compared to that from uninfected mice (y axis). Genes up-regulated in AKR and/or BALB/c gut tissue are labeled by gene symbol: angiogenin-like (Angl), pancreatitis-associated protein (Pap), mouse mast cell protease 1 (Mcpt1), indoleamine-2,3-dioxygenase (Indo), phospholipase A2 group IIA (Pla2g2a), mucin 2 (Muc2), von Hippel-Lindau binding protein 1 (Vbp1), nonagouti (a), chloride channel calcium-activated 3 (Clca3), T-cell-specific GTPase (Tgtp), desmoyokin (Ahnak), K-large-conductance calcium (Kcnmb1), and phosphatidylinositol membrane associated (Pitnm). Genes regulated by an average log₂-fold change of ≥+0.9 or ≤−0.9 have a false discovery rate at this threshold of 2.9% by SAM analysis (44). Gut tissue was pooled from five individual animals within a group, and two independent infection experiments were run. The average log₂-fold changes presented represent values calculated from both infection experiments.

FIG. 3.

Serum mast cell protease 1 protein levels precisely mirror changes in mRNA levels revealed by microarray. MMCP-1 levels in the sera of infected AKR and BALB/c mice at day 19 and day 60 postinfection compared to uninfected (naïve) levels are shown. Significantly higher levels of MMCP-1 were seen in BALB/c mice at day 19 postinfection (**P < 0.001) than in AKR mice at this time point, with levels declining by day 60 postinfection. Levels of MMCP-1 in AKR mice show a gradual rise postinfection. Levels are means ± standard deviations for five mice per time point.

FIG. 4.

Semiquantitative RT-PCR confirms the mRNA expression profiles of indoleamine-1,2-dioxygenase, intelectin, CXCL9, and CXCL11 as revealed by microarray. RT-PCR analyses of the expression levels of (A) indoleamine-1,2-dioxygenase (Indo), (B) CXCL11, (C) CXCL9, and (D) intelectin (Itln) at day 19 postinfection in gut tissue from AKR and BALB/c mice are shown. cDNA for HPRT was titrated out, and nonsaturated dilutions giving bands of equivalent brightness, shown as a single band per sample in the figure, were selected as the starting dilution for the serial dilutions. cDNA was serially diluted (1:2) and amplified by PCR as described in Materials and Methods. A, AKR; B, BALB/c. N, naïve; 19, day 19 postinfection.

FIG. 5.

RT-PCR detects an up-regulation in gamma interferon transcripts postinfection that is not detected by microarray. (A and B) Gamma interferon expression in whole gut tissue from (A) individual infected AKR mice at day 19 postinfection (lanes 1 to 3) and naïve AKR mice (lanes 4 to 8) and (B) infected BALB/c mice (lanes 1 to 2) and naïve BALB/c mice (lanes 3 to 4). (C) Gamma interferon expression in whole gut tissue using the same pools of tissue from infected and naïve AKR and BALB/c mice as those used for microarray analysis. Lane 1, infected AKR; lane 2, naïve AKR; lane 3, infected BALB/c; lane 4, naïve BALB/c. The starting amount of cDNA was determined by PCR for HPRT.

FIG. 6.

Epithelial cells are a source of indoleamine-2,3-dioxygenase in susceptible mice and intelectin in resistant mice. Indoleamine-2,3-dioxygenase (A) and intelectin (B) expression in intestinal epithelial cells was measured by RT-PCR. Epithelial cells were isolated from the large intestine of naïve or T. muris-infected AKR and BALB/c mice, and total RNA was extracted. cDNA for HPRT was titrated out, and nonsaturated dilutions giving bands of equivalent brightness, shown as a single band per sample in the figure, were selected as the starting dilution for the serial dilutions. cDNA was serially diluted (1:2) and amplified by PCR as described in Materials and Methods. A, AKR; B, BALB/c. N, naïve; 21, day 21 postinfection.

FIG. 7.

Goblet cell hyperplasia correlates with worm expulsion in resistant mice. Goblet cell hyperplasia in AKR and BALB/c mice at day 22 and day 34 postinfection were compared to uninfected (naïve) levels. Values are means ± standard deviations for mice per time point from an experiment run separately from the microarray analyses. Goblet cell numbers are significantly elevated (*P < 0.05) in BALB/c mice at day 22 postinfection compared to those of AKR mice, a time when transcripts for three antimicrobial proteins (intelectin, angiogenin-like protein, and angiogenin-related protein) show large severalfold increases in this mouse strain.