Broad up-regulation of innate defense factors during acute cholera

Abstract

We used a whole-genome microarray screening system (Affymetrix human GeneChips covering 47,000 different transcripts) to examine the gene expression in duodenal mucosa during acute cholera. Biopsies were taken from the duodenal mucosa of seven cholera patients 2 and 30 days after the onset of diarrhea, and the gene expression patterns in the acute- and convalescent-phase samples were compared pairwise. Of about 21,000 transcripts expressed in the intestinal epithelium, 29 were defined as transcripts that were up-regulated and 33 were defined as transcripts that were down-regulated during acute cholera. The majority of the up-regulated genes characterized were found to have an established or possible role in the innate defense against infections; these genes included the LPLUNC1, LF, VCC1, TCN1, CD55, SERPINA3, MMP1, MMP3, IL1B, LCN2, SOCS3, GDF15, SLPI, CXCL13, and MUC1 genes. The results of confirmative PCR correlated well with the microarray data. An immunohistochemical analysis revealed increased expression of lactoferrin in lamina propria cells and increased expression of CD55 in epithelial cells, whereas increased expression of the SERPINA3 protein (alpha1-antichymotrypsin) was detected in both lamina propria and epithelial cells during acute cholera. The expression pattern of CD55 and SERPINA3 in cholera toxin (CT)-stimulated Caco-2 cells was the same as the pattern found in the intestinal mucosa during acute cholera, indicating that the activation of the CD55 and SERPINA3 genes in intestinal epithelium was induced by CT. In conclusion, during acute cholera infection, innate defense mechanisms are switched on to an extent not described previously. Both direct effects of CT on the epithelial cells and changes in the lamina propria cells contribute to this up-regulation.

FIG. 1.

Differentially expressed genes during acute cholera compared to convalescence, expressed as log₂ ratios determined by RT-PCR. The horizontal lines indicate means. The circles represent measurements for patients with (filled circles) and without (open circles) up-regulation, as determined by the microarray technique using an acute-phase/convalescent-phase log₂ ratio of >0.5 to define individual up-regulation. The dashed line indicates the individual threshold value used during the microarray screening.

FIG. 2.

Expression of the CD55 and SERPINA3 genes in Caco-2 cells stimulated with CT for 18 h. The relative expression values are adjusted log₂ transcript abundance ratios of the target gene to the glyceraldehyde-3-phosphate dehydrogenase gene, as determined by RT-PCR. Means are indicated by horizontal lines. Three asterisks indicate that there is a significant difference between the CT-challenged group and the control (P < 0.001).

FIG. 3.

Immunolocalization of lactoferrin, CD55, and α₁-antichymotrypsin in duodenal biopsies during acute- and convalescent-phase cholera. The arrows indicate CD55 expression in the crypt epithelium.

FIG. 4.

Differential localization of CD8⁺ cells in duodenal biopsies during acute-phase (upper panel) and convalescent-phase (lower panel) cholera as revealed by immunohistochemical analysis.