447 - Proteomic Analysis of Mechanisms Underlying the Preconditioning Effect: Toward Improving Outcomes in Preterm Infants Exposed to Intrauterine Inflammation

Publication Number: 2435.447

Kazumasa Fuwa, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan; CHIHIRO MUKAI, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan; Hidetoshi Go, Nihon University School of Medicine, 30-1 Oyaguchi Kamicho Itabashi, Tokyo, Japan; Nobuhiko Nagano, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan; Ichiro Morioka, Nihon University School of Medicine, Itabashi, Tokyo, Japan

Background: Neonates born to pregnant mice pre-exposed to lipopolysaccharide (LPS) show improved survival upon subsequent challenge with LPS after birth, demonstrating a preconditioning effect. This phenomenon possibly contributes to the improved survival of preterm infants exposed to intrauterine inflammation.

Objective: To elucidate the molecular mechanisms underlying the preconditioning effect via proteomic analysis in a murine model.

Design/Methods: Pregnant mice were intraperitoneally injected with LPS (4 μg/dose) or saline on gestational days 16 and 17. The number, sex, and body weight of pups were recorded. Neonates received intraperitoneal injections of LPS (3 mg/kg) or saline on postnatal days (P)-3 and -5 (Figure 1). The following four groups were studied: Saline during pregnancy-saline after birth (S-S; n=4 dams → 49 pups), saline-LPS (S-L; n=6 dams → 69 pups), LPS-saline (L-S; n=6 dams → 44 pups), and LPS-LPS (L-L; n=10 dams → 64 pups). Survival was assessed on P6 based on the birth number, and blood samples were collected on P6 and P20. Proteomic profiling of samples from six males and six females (three pups from different dams per group) in the S-L and L-L groups was performed using the Olink Target 48 Mouse Cytokine panel.

Results: Preterm birth rate was higher in LPS-treated mothers (50%) than in saline-treated mothers (10%; p=0.0873). The mean litter size was significantly lower in the LPS-treated mothers (6.8±4.3 vs. 11.8±1.7; p< 0.05). From P3 to P6, L-L pups of both sexes showed persistently lower body weights than those in the other groups. Survival rates on P6 were as follows: S-S = 100%, S-L = 62.5%, L-S = 100%, and L-L = 92.9% in males; S-S = 100%, S-L = 70.8%, L-S = 94.1%, and L-L = 88.8% in females. Results of proteomic analysis during the postnatal period are presented in Tables 1 and 2. Proteomic analysis revealed that acute inflammatory cytokine (interleukin [IL]-6, IL-1β, tumor necrosis factor, and colony-stimulating factor-3) levels were decreased in the L-L group on P6. In males, early induction of regulatory T-cell-related cytokines (IL-27 and IL-2) and tissue repair-associated IL-22 was observed. On P20, females showed decreased IL-27 levels and relative dominance of Th17 cytokines (IL-17A and IL-17F).

Conclusion(s): In both sexes, L-L group showed higher survival than the S-L group on P6, although a persistent trend toward decreased body weight was observed. Overall, our proteomic analysis suggests that the preconditioning effect is associated with the accelerated resolution of acute inflammation and early induction of regulatory T-cell responses.

Experimental protocol
Abbreviations: LPS, lipopolysaccharide; i.p., intraperitoneal; GD, gestational day; P, postnatal day.

Results of the Proteomic Analysis at Postnatal Day 6


Results of the Proteomic Analysis at Postnatal Day 20