Focused examination of the intestinal epithelium reveals transcriptional signatures consistent with disturbances in enterocyte maturation and differentiation during the course of SIV infection

Abstract

The Gastrointestinal (GI) tract plays a pivotal role in AIDS pathogenesis as it is the primary site for viral transmission, replication and CD4(+) T cell destruction. Accordingly, GI disease (enteropathy) has become a well-known complication and a driver of AIDS progression. To better understand the molecular mechanisms underlying GI disease we analyzed global gene expression profiles sequentially in the intestinal epithelium of the same animals before SIV infection and at 21 and 90 days post infection (DPI). More importantly we obtained sequential excisional intestinal biopsies and examined distinct mucosal components (epithelium. intraepithelial lymphocytes, lamina propria lymphocytes, fibrovascular stroma) separately. Here we report data pertaining to the epithelium. Overall genes associated with epithelial cell renewal/proliferation/differentiation, permeability and adhesion were significantly down regulated (<1.5-7 fold) at 21 and 90DPI. Genes regulating focal adhesions (n = 6), gap junctions (n = 3), ErbB (n = 3) and Wnt signaling (n = 4) were markedly down at 21DPI and the number of genes in each of these groups that were down regulated doubled between 21 and 90DPI. Notable genes included FAK, ITGA6, PDGF, TGFβ3, Ezrin, FZD6, WNT10A, and TCF7L2. In addition, at 90DPI genes regulating ECM-receptor interactions (laminins and ITGB1), epithelial cell gene expression (PDX1, KLF6), polarity/tight junction formation (PARD3B&6B) and histone demethylase (JMJD3) were also down regulated. In contrast, expression of NOTCH3, notch target genes (HES4, HES7) and EZH2 (histone methyltransferase) were significantly increased at 90DPI. The altered expression of genes linked to Wnt signaling together with decreased expression of PDX1, PARD3B, PARD6B and SDK1 suggests marked perturbations in intestinal epithelial function and homeostasis leading to breakdown of the mucosal barrier. More importantly, the divergent expression patterns of EZH2 and JMJD3 suggests that an epigenetic mechanism involving histone modifications may contribute to the massive decrease in gene expression at 90DPI leading to defects in enterocyte maturation and differentiation.

Figure 1. SIV infection results in elevated plasma viremia and rapid depletion of mucosal CD4+ T cells.

Plasma viral loads (A) and changes in mucosal CD4+ T cells (B) “memory” CD4⁺ T cells (CD45RAneg, CCR5+) (C), and mucosal CD8⁺ T cells (D) in three Indian origin rhesus macaques at 21 and 90 days after intravenous infection with SIVmac251. Changes in CD45RA+/CCR5- populations at the 21 and 90d timepoints were not statistically significant (p>0.05).

Figure 2. Gene functional categories up (1.5 fold) in the intestinal epithelium at 21DPI.

The relative size of each sector in the pie chart is determined by the number of genes in that functional category. Genes with unknown function are not included in the pie chart. Only a few transcripts of importance to SIV infection are shown in the figure under each functional category. The full list of genes grouped under each functional category for the 21DPI time point is provided in supplementary table S1.

Figure 3. Gene functional categories down (1.5 fold) in the intestinal epithelium at 21DPI.

The relative size of each sector in the pie chart is determined by the number of genes in that functional category. Genes with unknown function are not included in the pie chart. Only a few transcripts of importance to SIV infection are shown in the figure under each functional category. The full list of genes grouped under each functional category for the 21DPI time point is provided in supplementary table S1.

Figure 4. Gene functional categories up (1.5 fold) in the intestinal epithelium at 90DPI.

The relative size of each sector in the pie chart is determined by the number of genes in that functional category. Genes with unknown function are not included in the pie chart. Only a few transcripts of importance to SIV infection are shown in the figure under each functional category. The full list of genes grouped under each functional category for the 90d time point is provided in supplementary table S2.

Figure 5. Gene functional categories down (1.5 fold) in the intestinal epithelium at 90DPI.

The relative size of each sector in the pie chart is determined by the number of genes in that functional category. Genes with unknown function are not included in the pie chart. Only a few transcripts of importance to SIV infection are shown in the figure under each functional category. The full list of genes grouped under each functional category for the 90d time point is provided in supplementary table S2.

Figure 6. FAK and TCF7L2 expression is significantly decreased in the intestinal epithelium during acute and chronic SIV infection.

Relative abundance in gene expression for FAK (light bars) and TCF7L2 (dark bars) in the intestinal epithelial cellular compartment of the jejunum at 21DPI (n = 4) and 6 months (n = 6) post SIV infection detected using quantitative real-time SYBR green two-step RT-PCR. The fold differences in gene expression were calculated as described in Materials and Methods. The relative fold increase is shown adjacent to each bar graph. The asterisk (*) indicates statistical significance (p<0.05).