Interferon-gamma expression by intraepithelial lymphocytes results in a loss of epithelial barrier function in a mouse model of total parenteral nutrition

Abstract

Objective: To determine the etiology of the loss of epithelial barrier function observed with the administration of total parenteral nutrition (TPN) in a mouse model.

Summary background data: Removal of enteral nutrition with the administration of TPN is associated with a loss of intestinal epithelial barrier function. The etiology of this barrier loss is not clear. Because intraepithelial lymphocytes (IELs) produce a number of cytokines that may alter epithelial permeability, the authors investigated IEL cytokine expression in a mouse model of TPN.

Methods: Adult C57BL/6 mice received TPN or enteral diet for 7 days. IELs were subsequently harvested and the mRNA expression of cytokines was measured. Epithelial barrier function was assessed in vitro with 51Cr-EDTA in Ussing chambers and was expressed as the permeability coefficient (Papp).

Results: IEL mRNA expression of interferon-gamma (IFN-gamma) rose from 0.14 +/- 0.07 in the control (enterally fed) group to 0.44 +/- 0.11 attomoles/microL in the TPN group (P <.05). Transforming growth factor-beta1 declined slightly but not significantly, from 0.75 +/- 0.35 to 0.55 +/- 0.18 attomoles/microL in the control and TPN groups, respectively. Epithelial barrier function declined significantly with TPN compared to controls. To assess the relevance of IFN-gamma changes, permeability in IFN-gamma knockout mice was studied. Barrier function was significantly higher in IFN-gamma knockout mice on TPN compared to C57BL/6 mice that received TPN.

Conclusions: IEL cytokine expression changes significantly with TPN administration. The partial correction with IFN-gamma knockout mice suggests that an upregulation of IFN-gamma expression is one mechanism responsible for the loss of the epithelial barrier associated with TPN.

Figure 1. Quantitative expression of interferon gamma (IFN-γ) and transforming growth factor-β1 (TGF-β) mRNA. RNA was isolated from the intestinal mucosa of control mice and mice on TPN (n = 8 per group). *P < .05.

Figure 2. Quantitative expression of interferon gamma (IFN-γ) (A) and transforming growth factor-β1 (TGF-β) (B) mRNA, as isolated from the intestinal mucosa of control mice and mice on TPN (n = 6 per group). Quantitative analysis was done after the cells in the intestinal mucosa were biomagnetically separated into CD45+ and CD45- groups as markers of lymphoid (IEL) and nonlymphoid (e.g., epithelial cells) populations, respectively. *P < .05 and **P < .01, comparing cytokine expression between lymphoid (IEL) and nonlymphoid (epithelial cell) populations.

Figure 3. Quantitative expression of interferon gamma (IFN-γ) (A) and transforming growth factor-β1 (TGF-β) (B) mRNA, as isolated from the intestinal mucosa of control mice and mice on TPN (n = 6 per group). Quantitative analysis was done after the cells in the intestinal mucosa were biomagnetically separated into CD8αα+ and CD8αβ+ groups. *P < .05 and **P < .01 comparing cytokine expression between thymic-independent (CD8αα+) and thymic-dependent (CD8αβ+) populations.

Figure 4. Results of intracellular staining of IFN-γ. The gated subpopulations IEL are shown for both control and TPN groups (n = 6 per group). The percentages of each quadrant are shown, with the right upper quadrant representing IFN-γ-positive cells in the respective cell population. See the “Results” section for the mean percentage of IFN-γ expression in individual cell populations.

Figure 5. Results of intestinal permeability as measured by the permeability coefficient (Papp) from Ussing chamber experiments (n = 8–10 per group). Study of epithelial barrier function in both C57BL/6J and IFN-γ knockout mice after a 7-day period of enteral nutrition or TPN (P < .05 between groups). Epithelial leak is expressed as the Papp of ⁵¹Cr-EDTA. *P < .05 compared with control. The IFN-γ knockout TPN group permeability was also significantly (P < .05) lower than the wild-type TPN mouse group.