Chorioamnionitis-induced fetal gut injury is mediated by direct gut exposure of inflammatory mediators or by lung inflammation

Abstract

Intra-amniotic exposure to proinflammatory agonists causes chorioamnionitis and fetal gut inflammation. Fetal gut inflammation is associated with mucosal injury and impaired gut development. We tested whether this detrimental inflammatory response of the fetal gut results from a direct local (gut derived) or an indirect inflammatory response mediated by the chorioamnion/skin or lung, since these organs are also in direct contact with the amniotic fluid. The gastrointestinal tract was isolated from the respiratory tract and the amnion/skin epithelia by fetal surgery in time-mated ewes. Lipopolysaccharide (LPS) or saline (controls) was selectively infused in the gastrointestinal tract, trachea, or amniotic compartment at 2 or 6 days before preterm delivery at 124 days gestation (term 150 days). Gastrointestinal and intratracheal LPS exposure caused distinct inflammatory responses in the fetal gut. Inflammatory responses could be distinguished by the influx of leukocytes (MPO(+), CD3(+), and FoxP3(+) cells), tumor necrosis factor-α, and interferon-γ expression and differential upregulation of mRNA levels for Toll-like receptor 1, 2, 4, and 6. Fetal gut inflammation after direct intestinal LPS exposure resulted in severe loss of the tight junctional protein zonula occludens protein 1 (ZO-1) and increased mitosis of intestinal epithelial cells. Inflammation of the fetal gut after selective LPS instillation in the lungs caused only mild disruption of ZO-1, loss in epithelial cell integrity, and impaired epithelial differentiation. LPS exposure of the amnion/skin epithelia did not result in gut inflammation or morphological, structural, and functional changes. Our results indicate that the detrimental consequences of chorioamnionitis on fetal gut development are the combined result of local gut and lung-mediated inflammatory responses.

Keywords: endotoxin; fetal inflammatory response; necrotizing enterocolitis; sheep.

Fig. 1.

The concentration of circulating haptoglobin was measured by enzyme-linked immunosorbent assay (ELISA), and groups were compared with the Mann-Whitney test. Haptoglobin plasma levels significantly increased in the 2-day “IA Ocln,” 2-day “gut,” and the 2-day “lung” LPS groups (A). No significant changes in circulating haptoglobin levels were detected after 6 days of lipopolysaccharide (LPS) exposure (B). IA, intra-amniotic; Ocln,. *P < 0.05 vs. controls using a Mann-Whitney nonparametric U-test.

Fig. 2.

For each experimental group, myeloperoxidase (MPO, A and B)-, CD3 (C and D)-, or FoxP3 (E and F)-positive cells were counted in the fetal ileum per high-power field, and the average value of the sum of 3 representative areas is given. Groups were compared with the Mann-Whitney test. *P < 0.05 vs. control using a Mann-Whitney nonparametric U-test.

Fig. 3.

Quantitative real-time PCR assays in the fetal ileum using ovine-specific primers and Taqman probes. The values for tumor necrosis factor (TNF)-α (A and B), IL-1β (C and D), IL-6 (E and F), and interferon (IFN)-γ (G and H) were normalized to 18S rRNA, and groups were compared with the Mann-Whitney test. The mean mRNA signal in controls was assigned to a value of 1. Mean fold changes in mRNA expression of all other experimental groups were expressed relative to controls. *P < 0.05 vs. control using a Mann-Whitney nonparametric U-test.

Fig. 4.

Quantitative real-time PCR mRNA expression levels of Toll-like receptors (TLRs) in the fetal ileum using sheep-specific primers and Taqman probes. TLR mRNA values were normalized to 18S rRNA, and groups were compared with the Mann-Whitney test. The mean mRNA signal in controls was set to 1, and levels at each time point were expressed in a relative manner. *P < 0.05 vs. control using a Mann-Whitney nonparametric U-test.

Fig. 5.

For each experimental group, the ileal distribution of Kruppel-like factor 5-positive (KLF5⁺) cells was analyzed by immunohistochemistry. Compared with control animals (A), reduced KLF5⁺ cell numbers were observed in fetuses of the “2-day LPS lung” group (E). The distribution of KLF5⁺ cells in the “2-day LPS IA” (B), “2-day LPS Ocln” (C), “2-day LPS gut” (D), “6-day LPS IA” (F), “6-day LPS Ocln” (G), “6-day LPS gut” (H), and “6-day LPS lung” (I) LPS groups did not change. Magnification ×200.

Fig. 6.

The numbers of proliferating and mitotic cells in the fetal ileum were measured by an immunohistochemical staining for Ki67 and phospho-histone H3 (pHistoneH3), respectively. Gut exposure to LPS for 2 days increased the number of Ki67- and phospho-histone H3-positive cells (A and B). Compared with controls, intra-amniotic LPS exposure for 6 days resulted in increased Ki67 and phospho-histone H3-positive cells (C and D). Groups were compared with the Mann-Whitney test. *P < 0.05 vs. control using a Mann-Whitney nonparametric U-test.

Fig. 7.

Zonula occludens protein 1 (ZO-1) distribution was assessed in fetal ileal tissue using an immunofluorescent staining. A fragmented ZO-1 staining was observed in premature saline-treated control animals (A) and in lambs from the “2-day IA LPS” (B), 2-day LPS Ocln (C), 2-day LPS gut (D), or 2-day LPS lung (E) groups. ZO-1 distribution was even more disturbed in the 6-day LPS IA (F), 6-day LPS gut (H), and 6-day LPS lung (I) groups. The ZO-1 distribution pattern in the 6-day LPS Ocln group (G) was comparable to saline-treated control animals (A). Magnification ×200.

Fig. 8.

Circulating intestinal fatty acid-binding protein (I-FABP) levels were measured by ELISA, and groups were compared with the Mann-Whitney test. I-FABP plasma levels did not change after exposure to LPS for 2 days in each experimental group (A). Lung exposure to LPS for 6 days significantly increased I-FABP plasma levels (B). *P < 0.05 vs. control using a Mann-Whitney nonparametric U-test.