Vitamin A Supplementation and Lipopolysaccharide‐Induced Inflammation in the Neonate — A Pilot Study in C57BL/6 Mice
Authors: Wei Cheng‐Hsin , Ross A. Catharine 
Published: November 2023

Vitamin A (VA) is important in host defense against infections. Previous studies have suggested that retinoic acid, the active metabolite of VA, is critical in mucosal immunity and immune tolerance. However, studies are limited about how VA supplementation affects mucosal immune responses to inflammation in neonates. The objective of the study was to investigate the interactions between micronutrient (i.e., VA supplementation) and inflammation during the neonatal period, when intestinal epithelial crypts are formed and immune cell populations rapidly increase. We hypothesized there would be both independent effects of VA supplementation and lipopolysaccharide (LPS) treatment, as well as a potential modifying effect of VA supplementation on the LPS‐induced inflammatory response in neonates. A purified AIN‐93 VA‐marginal diet, containing 0.35 μg of retinol as retinyl palmitate per g diet, was fed to C57BL/6 dams from pregnancy until their pups (n=16) were euthanized on postnatal day (P) 14. Two doses of VA, 6 mg/kg of body weight, or canola oil as control, were administered orally on P12 and P13. LPS, 2 mg/kg of body weight, or phosphate‐buffered saline (PBS) as control, was injected intraperitoneally 3 h after the second dose of VA or oil on P13 to induce inflammation. Plasma and tissues were collected 18 h after LPS injection on P14. The small intestine samples were divided into upper and lower sections to determine differences between intestinal sections. Tissue total retinoid concentrations were determined by Ultra Performance Liquid Chromatography (UPLC). Cytokine analyses, including IFN‐γ, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, KC/GRO, IL‐10, IL12p70, and TNF‐α, were performed on plasma and intestinal tissue homogenates using V‐PLEX validated assay kits. For statistical analysis, two‐way ANOVA was performed to examine two independent factors (i.e., VA supplementation and LPS‐induced inflammation), as well as their interactions. Differences among treatment groups were determined by Tukey's multiple comparison test. Pups with LPS‐induced inflammation lost 7.5% of body weight and had reduced stomach weight within the 18‐h period after LPS injection, while control pups gained 10.8% body weight. Total retinol concentrations in tissues, including liver, lung, and upper and lower small intestine, were significantly higher in VA‐supplemented as compared to oil‐treated pups. However, no significant difference was observed on tissue retinoid storage between LPS‐treated and control pups. Serum cytokine concentrations, including IFN‐γ, IL‐4, IL‐6, KC/GRO, IL‐10, IL12p70, and TNF‐α, were significantly higher in LPS‐treated pups regardless of VA treatment. Overall, cytokine concentrations were much higher in the intestine as compared to in serum, other than for IL‐10. For both upper and lower small intestine, the concentrations of IL‐1β and KC/GRO were higher in LPS‐treated pups and, interestingly, even higher in the VA‐supplemented pups under LPS‐induced inflammation. The results indicated that VA supplementation and LPS exert differential effects, but VA might also affect intestinal immune responses to LPS‐induced inflammation in neonates. Future studies are warranted to further investigate these interactions.