Lipopolysaccharide-induced injury is more pronounced in fetal transgenic ErbB4-deleted lungs

Abstract

Pulmonary ErbB4 deletion leads to a delay in fetal lung development, alveolar simplification, and lung function disturbances in adult mice. We generated a model of intrauterine infection in ErbB4 transgenic mice to study the additive effects of antenatal LPS administration and ErbB4 deletion during fetal lung development. Pregnant mice were treated intra-amniotically with an LPS dose of 4 μg at E17 of gestation. Lungs were analyzed 24 h later. A significant influx of inflammatory cells was seen in all LPS-treated lungs. In heterozygote control lungs, LPS treatment resulted in a delay of lung morphogenesis characterized by a significant increase in the fraction of mesenchyme, a decrease in gas exchange area, and disorganization of elastic fibers. Surfactant protein (Sftp)b and Sftpc were upregulated, but mRNA of Sftpb and Sftpc was downregulated compared with non-LPS-treated controls. The mRNA of Sftpa1 and Sftpd was upregulated. In ErbB4-deleted lungs, the LPS effects were more pronounced, resulting in a further delay in morphological development, a more pronounced inflammation in the parenchyma, and a significant higher increase in all Sftp. The effect on Sftpb and Sftpc mRNA was somewhat different, resulting in a significant increase. These results imply a major role of ErbB4 in LPS-induced signaling in structural and functional lung development.

Fig. 1.

LPS affects the influx of inflammatory cells to the chorioamnion. Inflammatory cells (arrows) in the chorioamnion (1) of normal saline (NS)-treated HER4^heart+/− (A) and HER4^heart−/− (C) control animals and LPS-treated HER4^heart+/− (B) and HER4^heart−/− (D) animals.

Fig. 2.

LPS potentiates the delay in morphological development of ErbB4-deleted lungs (histology). Frozen lung sections were stained with hematoxylin and eosin. Lung parenchyma of fetal E18 lungs from control HER4^heart+/− (A and B) and ErbB4-deleted HER4^heart−/− [C and D; E (higher magnification)] animals, 24 h after intra-amniotic injection of NS (controls) (A and C) or LPS [B and D; E (higher magnification)]. In the higher magnification, mesenchymal cells (arrows) as well as neutrophils granulocytes (arrowheads) are seen in the lung parenchyma of LPS-injected ErbB4-deleted HER4^heart−/− lungs (E).

Fig. 3.

LPS potentiates the delay in morphological development of ErbB4-deleted lungs (stereological analysis). Lungs were harvested 24 h after intra-amniotic NS (white bars for ^+/−, light gray bars for ^−/−) and LPS (dark gray bars for ^+/−, black bars for ^−/−) injection from heterozygote control HER4^heart+/− and ErbB4-deleted HER4^heart−/− animals. Measurement of the volume density (V_v) of the mesenchyme serves as an indicator for pulmonary immaturity (A), the V_v of air spaces reflects the opening of air containing sacculi (B), the V_v of the alveolar septa reflects the maturity of lungs (C), and the surface density (S_v) of the alveolar septa determines the maturity of the gas exchange area (D). Values are given as means ± SE, *P < 0.05.

Fig. 4.

LPS induces influx of inflammatory cells and expression of TNF-α in ErbB4-deleted lungs. CD11b staining of inflammatory cells (arrows) in lung sections 24 h after intra-amniotic injection of NS (A) or LPS (C) in control HER4^heart+/− and NS (B) or LPS (D) in ErbB4-deleted HER4^heart−/− animals. Granulocyte (E) and macrophage (F) numbers are presented as cell density (cells/mm²) 24 h after intra-amniotic injection of NS (bars: light gray for ^+/−, gray for ^−/−) and LPS (bars: dark gray for ^+/−, black for ^−/−) in heterozygote control HER4^heart+/− and ErbB4-deleted HER4^heart−/− animals. TNF-α concentrations were measured by ELISA (G). The data of LPS-treated (solid bars) lungs from heterozygote control HER4^heart+/− (right) and ErbB4-deleted HER4^heart−/− (left) animals are given as relative percentages of the NS-treated sham controls, whose TNF-α values were set as 100%. Data are shown as means ± SE, *P < 0.05.

Fig. 5.

LPS potentiates the reduction and disorganization of elastic fibers in ErbB4-deleted fetal lungs. Staining of elastic fibers (arrows) with orcein 24 h after intra-amniotic injection of NS (A) or LPS (C) in control HER4^heart+/− and NS (B) or LPS (D) in ErbB4-deleted HER4^heart−/− lungs.

Fig. 6.

LPS potentiates the ErbB4 deletion-induced changes in Sftp (SP) and Sftp mRNA expression. A: Sftpa1, Sftpb, Sftpc, and Sftpd expression in LPS-treated heterozygote control HER4^heart+/− (dark gray bars) and ErbB4-deleted HER4^heart−/− (light gray bars) lungs. Surfactant protein (SP) content was quantified by densitometry, and data are presented as relative percentages of NS-treated control lungs, whose values were adapted to 100%. B: mRNA expression of Sftpa1, Sftpb, Sftpc, and Sftpd in LPS-treated heterozygote control HER4^heart+/− (dark gray bars) and ErbB4-deleted HER4^heart−/− (light gray bars) lungs. Data are presented as the differences in the threshold cycles (DCt) of Sftp to the actin gene as a housekeeping gene. DCt values are inversely proportional to the levels of mRNA. DCt values of the ErbB4-deleted HER4^heart−/− animals were expressed as DDCt of their litter-specific heterozygote control HER4^heart+/− lungs. Values are shown as means ± SE. **P < 0.01 compared with NS-treated controls; +P < 0.05 and ++P < 0.01, comparing LPS-treated HER4^heart+/− and HER4^heart−/− fetal lungs.