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. 2004 Oct;72(10):6076-86.
doi: 10.1128/IAI.72.10.6076-6086.2004.

Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis

Pamela A Knight  1 , Alan D Pemberton, Kevin A Robertson, Douglas J Roy, Steven H Wright, Hugh R P Miller
Affiliations

Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis

Pamela A Knight et al. Infect Immun. 2004 Oct.

Abstract

Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule beta (RELMbeta) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmbeta and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmbeta).

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Figures

FIG. 1.
FIG. 1.
Clustering of differentially expressed genes. To investigate the similarity of samples in this experiment, hierarchical clustering (using Pearson correlation as a distance measure) of a filtered subset of 1,662 variable genes (coefficient of variation > 0.4) was undertaken in GeneSpring (Silicon Genetics). The dendrogram relates samples by their gene expression pattern with short branch length, indicating similarity. Each column represents one sample, and each row represents a gene. Biological replicates (uninfected, red branches; infected, green branches) cluster together. To aid in visualization of the data, gene expression is shown as a color representation of the Z-score (see Materials and Methods for a description of data analysis). Green represents a decrease in expression from the mean value, whereas red represents an increase.
FIG. 2.
FIG. 2.
Gene expression in jejunal epithelium at day 0 and day 14 of T. spiralis infection. Preparation of epithelium, extraction of mRNA and microarray procedures are described in Materials and Methods. The mean day 0 signal (n = 4) is plotted against mean day 14 signal (n = 4) for each gene probe set present on the Affymetrix MG-U74AV2 array. Diagonal lines indicate the 10-fold level of up- and downregulation. The altered expression of selected transcripts is confirmed by separate RT-PCR analysis with specific primers (see Materials and Methods). Transcripts for Sprr2A, Mcpt-1 and Mcpt-2, Relmβ, and Siat4c are clearly upregulated. Transcripts for DNase I and Reg3α and -γ are downregulated, whereas mitochondrial creatine kinase (Ckmt1), a highly expressed housekeeping gene, is unchanged.
FIG. 3.
FIG. 3.
Temporal changes in expression of specific transcripts in jejunal epithelium after T. spiralis infection. (a) RT-PCR products for Mcpt-1 and Mcpt-2; Reg3α, -β, and -γ; Sprr2A; DNase I; Siat4c; Daf1; and Relmβ; with mitochondrial creatine kinase (Ckmt1) as a positive control for the RT reactions. Total RNA for RT was extracted from epithelium isolated from T. spiralis-infected BALB/c mice on days 0, 1, 3, 7, 14, 28, and 56 p.i. (n = 4/time point). (b) Histograms showing relative net intensities of the RT-PCR products in panel a normalized against the highest net intensity recorded with each primer set to visualize changes in transcript levels over time. ✽, Significantly different from uninfected levels (P ≤ 0.05).
FIG. 4.
FIG. 4.
Change in expression of Sprrs by jejunal epithelium in response to T. spiralis infection. The hybridization intensities for Sprrs at days 0 and 14 of infection are compared (expressed as the mean ± the standard deviation [n = 4]). Upregulation of Sprr2A was observed (P ≤ 0.001), but no change was seen for Sprr1A, -1B, or -3. The signal cutoff level of 50 is shown as a broken line.
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