Intestinal epithelial cell-derived interleukin-7: A mechanism for the alteration of intraepithelial lymphocytes in a mouse model of total parenteral nutrition

Abstract

Total parenteral nutrition (TPN), with the absence of enteral nutrition, results in profound changes to both intestinal epithelial cells (EC) as well as the adjacent intraepithelial lymphocyte (IEL) population. Intestinal EC are a rich source of IL-7, a critical factor to support the maintenance of several lymphoid tissues, and TPN results in marked EC changes. On this basis, we hypothesized that TPN would diminish EC-derived IL-7 expression and that this would contribute to the observed changes in the IEL population. Mice received enteral food and intravenous crystalloid solution (control group) or TPN. TPN administration significantly decreased EC-derived IL-7 expression, along with significant changes in IEL phenotype; decreased IEL proliferation; and resulted in a marked decrease in IEL numbers. To better determine the relevance of TPN-related changes in IL-7, TPN mice supplemented with exogenous IL-7 or mice allowed ad libitum feeding and treated with exogenous administration of anti-IL-7 receptor (IL-7R) antibody were also studied. Exogenous IL-7 administration in TPN mice significantly attenuated TPN-associated IEL changes, whereas blocking IL-7R in normal mice resulted in several similar changes in IEL to those observed with TPN. These findings suggest that a decrease in EC-derived IL-7 expression may be a contributing mechanism to account for the observed TPN-associated IEL changes.

Figure 1

Changes in EC-derived IL-7 mRNA expression in intestinal mucosal specimens. Results are measured by real-time PCR, and are expressed as the ratio of the number of copies of the IL-7 gene to the number of copies of the β-actin gene multiplied by 10⁻⁵; n =6 per group. TPN administration led to a significant decrease in EC-derived IL-7 mRNA expression compared with controls *P<0.05.

Figure 2

Western immunoblot expression of IL-7 from isolated EC is shown. A. Composite representative immunoblot of EC-derived IL-7 expression in control and total parenteral nutrition (TPN) mice. B. TPN administration resulted in a significant decrease in EC-derived IL-7 expression. Results are the relative expression of IL-7 protein normalized to the expression of β-actin multiplied by 100; n =6 per group, *P<0.05 TPN compared to control group.

Figure 3

Representative flow cytometry results of gated IEL populations. Cell populations are expressed as the percentage of gated cells with CD8α and CD8β markers based on isotype-matched control antibodies. Data are expressed as the percent of total gated population of lymphocytes. TPN administration led to a loss of the CD8αβ+IEL subset. IL-7 administration attenuated the loss of CD8αβ+IEL associated with TPN administration (TPN+IL-7). Using anti-IL-7 receptor antibody (IL-7R blockade), led to a significant decrease in the CD8αβ+ IEL subset when compared with controls.

Figure 4

Distribution of the CD44+ IEL. CD44 was used as a marker of IEL maturation. TPN resulted in a significant decrease in the CD8+CD44+ IEL, P<0.05 compared to control mice. IL-7 administration in TPN treated mice attenuated the decrease in CD8+CD44+ and CD4+CD44+ IEL subsets when compared with the TPN group (P<0.05 compared to controls). Blocking IL-7R also resulted in a significant decrease in CD4+CD44+ and CD8+CD44+ sub-populations when compared with Controls (n =6 each group, *P<0.05).

Figure 5

Distribution of IEL phenotypes. TPN administration led to a loss of CD4+CD8+IEL and CD4+CD8− subsets. IL-7 administration significantly attenuated the loss of CD4+CD8+, and CD4+CD8− IEL sub-populations associated with TPN administration. Blocking IL-7R with anti-IL-7R antibody resulted in a significant decrease in both CD4+CD8− and CD4+CD8+ IEL compared to controls. (n =6 each group, *P<0.05).

Figure 6

TPN results in a decline in the proportion of IEL that are in in active cell proliferation. IEL were surface-labeled with fluorochrome-conjugated antibody to CD3, and then treated with propidium iodide for detection of cell cycle status using flow cytometric analysis. Histograms were generated by gating on IEL populations and displaying the cell cycle. TPN administration significantly decreased IEL proliferation when compared with Controls, P<0.05. Administration of IL-7 to TPN mice attenuated this decline, and IL-7R antibody replicated the decline in IEL proliferation. Results are the percentage of IEL in a proliferative phase (as determined by cells in the S/G2/M cell cycle phases). Results are the mean of n=6 mice in each group, *P<0.05 compared to the control group.

Figure 7

Composite gel demonstrating the expression of IL-7 receptor (IL-7R) mRNA in different IEL sub-populations. Samples are from representative specimens of various IEL sub-populations. Cells were purified using flow cytometry sorting. RT-PCR results demonstrated that CD4+, CD8αβ+, and CD8αα+ IEL sub-populations, as well as αβ-TCR+ and γδ TCR+ IEL subtypes express IL-7R mRNA. Results were consistently noted from at least three mice in each group.

Figure 8

Flow cytometric analysis of IL-7 receptor (IL-7R) expression in isolated IEL. Results are expressed as the mean percent of IL-7R+ cells in all gated IEL (n=6), with a minimum of 10,000 IEL analyzed per mouse. Note that the percentage of IL7R+ cells was greatest in the CD4+ and αβ-TCR+ IEL sub-populations, relative to the total number of cells in each of these subtypes.