IL-1α mediated chorioamnionitis induces depletion of FoxP3+ cells and ileal inflammation in the ovine fetal gut

Abstract

Background: Endotoxin induced chorioamnionitis increases IL-1 and provokes an inflammatory response in the fetal ileum that interferes with intestinal maturation. In the present study, we tested in an ovine chorioamnionitis model whether IL-1 is a major cytokine driving the inflammatory response in the fetal ileum.

Method: Sheep bearing singleton fetuses received a single intraamniotic injection of recombinant ovine IL-1α at 7, 3 or 1 d before caesarian delivery at 125 days gestational age (term = 150 days).

Results: 3 and 7 d after IL-1α administration, intestinal mRNA levels for IL-4, IL-10, IFN-γ and TNF-α were strongly elevated. Numbers of CD3+ and CD4+ T-lymphocytes and myeloidperoxidase+ cells were increased whereas FoxP3+ T-cells were detected at low frequency. This increased proinflammatory state was associated with ileal mucosal barrier loss as demonstrated by decreased levels of the intestinal fatty acid binding protein and disruption of the tight junctional protein ZO-1.

Conclusion: Intraamniotic IL-1α causes an acute detrimental inflammatory response in the ileum, suggesting that induction of IL-1 is a critical element in the pathophysiological effects of endotoxin induced chorioamnionitis. A disturbed balance between T-effector and FoxP3+ cells may contribute to this process.

Figure 1. Experimental design.

Antenatal inflammation was induced by a single injection of IL-1α under ultrasound guidance at 118, 122 or 124 d GA. Animals were delivered at 125 d GA and animals of the control group underwent the same procedure with an injection of saline.

Figure 2. Compared to control animals, a significant (*) increase of MPO immunoreactivity in the fetal terminal ileum was seen at 3 d and 7 d after IL-1α exposure.

For each experimental group, mean cell counts of MPO positive cells are given per high-power field (A). Representative sections of control animals and IL-1 treated animals for 3 d are depicted in B–C.

Figure 3. At 3 d and 7 d after intraamniotic IL-1α injection, significant increases of CD3 positive cells in the fetal terminal ileum were detected when compared with saline treated animals.

For each experimental group, mean cell counts of CD3 expressing cells per high-power field are depicted (A). Representative sections of saline and 3 d IL1α treated animals are shown (B–C).

Figure 4. At 3 d and 7 d after intraamniotic IL-1α injection, the number of CD4 positive cells significantly increased when compared with saline treated animals.

A) For each experimental group, mean cell counts of CD4 expressing cells are expressed per high-power field. B–C) Representative sections of saline and 3 d IL-1α treated animals are shown.

Figure 5. A single intraamniotic injection of IL-1α significantly reduced the number of FoxP3 expressing cells.

A) Mean cell counts of FoxP3+ positive cells were measured for the sum of 3 high-power fields. Representative ileal sections of saline (B) and 3 d IL1α (C) treated animals are shown. For inset, 400× magnification was used.

Figure 6. Quantification using real-time PCR assays using sheep specific primers and Taqman probes.

The values for each cytokine were normalized to 18s rRNA. The mean mRNA signal in control animals was given the value of 1 and levels at each time point were expressed relative to controls (*p<0.05 vs control).

Figure 7. The I-FABP content in the terminal ileum is reduced within 1 d after IL-1α treatment and I-FABP levels remain low up to 7 d after intraamniotic IL-1α administration.

A) Representative b-actin and I-FABP protein fragments are shown for each group. B-actin was used to confirm equal loading. Relative quantitative data were obtained by densitometric evaluation of actin and I-FABP products which were compared to a standard curve obtained by amplification of a serial dilution of a highly concentrated protein standard (*p<0.05 vs control).

Figure 8. The fragmented ZO-1 distribution in preterm control animals (A) was further disrupted in lambs exposed to IL-1α for 3 d (B) or 7 d (C).

Magnification 200×. For inset, 1000× magnification was used.