Identification of an IL-22-Dependent Gene Signature as a Pharmacodynamic Biomarker

Abstract

Interleukin-22 (IL-22) plays a role in epithelial barrier function and repair, and may provide benefits in conditions like inflammatory bowel disease. However, limited human data are available to assess the clinical effect of IL-22 administration. This study used a human intestinal cell line to identify an IL-22-dependent gene signature that could serve as a pharmacodynamic biomarker for IL-22 therapy. The response to IL-22Fc (UTTR1147A, an Fc-stabilized version of IL-22) was assessed in HT-29 cells by microarray, and the selected responsive genes were confirmed by qPCR. HT-29 cells demonstrated dose-dependent increases in STAT3 phosphorylation and multiple gene expression changes in response to UTTR1147A. Genes were selected that were upregulated by UTTR1147A, but to a lesser extent by IL-6, which also signals via STAT3. IL-1R1 was highly upregulated by UTTR1147A, and differential gene expression patterns were observed in response to IL-22Fc in the presence of IL-1β. An IL-22-dependent gene signature was identified that could serve as a pharmacodynamic biomarker in intestinal biopsies to support the clinical development of an IL-22 therapeutic. The differential gene expression pattern in the presence of IL-1β suggests that an inflammatory cytokine milieu in the disease setting could influence the clinical responses to IL-22.

Keywords: IBD; IL-1β; IL-22; antimicrobial; epithelial repair; gene expression; pharmacodynamic biomarker.

Figure 1

IL-22-mediated STAT3 activation, as measured in HT-29 cells. The HT-29 cells were cultured for 5 days with media, followed by an additional 18 h in serum-free media. Varying concentrations of IL-22 or 25 ng/mL rhIL-6 were added to the cell cultures for 20 min. The cell lysates were analyzed by Western blot. (A) Representative Western blot densitometry showing the pSTAT3 and total STAT3. (B) Levels of pSTAT3 normalized to STAT3, shown as the mean ± SD of three biological replicates. The EC₅₀ is 1.56 μg/mL.

Figure 2

Gene upregulation induced by IL-22 and not IL-6. (A) Scatterplot comparing the microarray gene expression from HT-29 cells cultured for 24 h with either UTTR1174A (IL-22Fc) or IL-6 prior to the RNA isolation. The colored points indicate genes with significant differential expression (2.0-fold change, adjusted p < 0.05). The labeled points indicate 13 genes with a fivefold change (adjusted p < 0.05). (B) Heat map from the same experiment using stricter specificity criteria, defined as the union of genes that were at least 1.5-fold upregulated by UTTR1147A, less than 1.2-fold upregulated by IL-6, and had at least a 1.5-fold difference between UTTR1147A and IL-6. Three biological replicates are represented.

Figure 3

Differential gene expression by IL-22 in the presence of IL-1β. (A) Heat map with the microarray gene expression from HT-29 cells cultured for 24 h with or without IL-1β, IL-22Fc or IL-22Fc + IL-1β prior to the RNA isolation. Three biological replicates are represented. (B) Scatterplot showing the genes upregulated by IL-22 alone vs. IL-22 + IL-1β.

Figure 4

IL-22 dose-responsive genes. (A) Representative plots of the genes that were the most dose responsive to IL-22Fc. (B) Representative plots of the genes that were the most dose responsive to IL-22Fc+ IL-1β.

Figure 5

Secretion of acute phase proteins by HT-29 cells requires IL-1β. (A) REG3A (Pancrepap), as measured by ELISA, showing detectable levels only with the addition of IL-1β, shown as the mean +/- SD, n = 2. The dashed red line indicates the LLOQ of the assay. (B) Serum Amyloid A (SAA), as measured by ELISA, showing detectable levels only with the addition of IL-1β, shown as the mean +/- SD, n = 2. The dashed red line indicates the LLOQ of the assay. (C) REG3A (Pancrepap) representative dose–response curve, as measured by ELISA. The points represent the mean of the duplicate wells, EC₅₀ = 2.3 μg/mL. (D) Serum Amyloid A (SAA) representative dose–response curve, as measured by ELISA. The points represent the mean of the duplicate wells, EC₅₀ = 0.74 μg/mL.