γ-Tocopherol supplementation of allergic female mice augments development of CD11c+CD11b+ dendritic cells in utero and allergic inflammation in neonates

Abstract

γ-Tocopherol increases responses to allergen challenge in allergic adult mice, but it is not known whether γ-tocopherol regulates the development of allergic disease. Development of allergic disease often occurs early in life. In clinical studies and animal models, offspring of allergic mothers have increased responsiveness to allergen challenge. Therefore, we determined whether γ-tocopherol augments development of allergic responses in offspring of allergic female mice. Allergic female mice were supplemented with γ-tocopherol starting at mating. The pups from allergic mothers developed allergic lung responses, whereas pups from saline-treated mothers did not respond to allergen challenge. The γ-tocopherol supplementation of allergic female mice increased the numbers of eosinophils twofold in the pup bronchoalveolar lavage and lungs after allergen challenge. There was also about a twofold increase in pup lung CD11b(+) subsets of CD11c(+) dendritic cells and in numbers of these dendritic cells expressing the transcription factor IRF4. There was no change in several CD11b(-) dendritic cell subsets. Furthermore, maternal supplementation with γ-tocopherol increased the number of fetal liver CD11b(+)CD11c(+) dendritic cells twofold in utero. In the pups, γ-tocopherol increased lung expression of the inflammatory mediators CCL11, amphiregulin, activin A, and IL-5. In conclusion, maternal supplementation with γ-tocopherol increased fetal development of subsets of dendritic cells that are critical for allergic responses and increased development of allergic responses in pups from allergic mothers. These results have implications for supplementation of allergic mothers with γ-tocopherol in prenatal vitamins.

Keywords: allergic lung inflammation; dendritic cells; vitamin E; γ-tocopherol.

Fig. 1.

Schematic of d-γ-tocopherol and d-α-tocopherol which differ by one methyl group (arrow).

Fig. 2.

Schematic of the timeline for d-α-tocopherol diet and ovalbumin (OVA) treatment of mothers and pups. Mothers were sensitized and challenged with OVA or saline, and then at the time of breeding, they were given basal diet or diet supplemented with 250 mg d-γ-tocopherol (d-γ-T)/kg of diet. On day 3 after birth, the pups were sensitized once with OVA/alum by intraperitoneal injection and then challenged with aerosolized 3% OVA on days 10–12. On postnatal day (PND) 13, the pup livers were analyzed for tocopherol isoforms by HPLC and the pup lungs were analyzed for bronchoalveolar lavage (BAL) inflammation and lung tissue dendritic cell (DC) subsets by multicolor flow cytometry. With another set of mothers, on gestational day (GD) 18, tocopherols were determined in the liver of the mothers by HPLC and the fetal liver was analyzed for DC subsets.

Fig. 3.

Mother and pup liver γ-tocopherol levels. Mice were treated as in Fig. 2. A: on GD18, concentrations of γ-tocopherol (γ-T) were determined in the liver of the mothers by HPLC. B: on PND13, the pup livers were analyzed for γ-tocopherol isoforms by HPLC. *P < 0.05, compared with all groups; n = 8−10 mice/group.

Fig. 4.

Maternal γ-tocopherol reduced the numbers of mated females that had pups but did not alter pup numbers per mother or pup body weight on day 13. Mice were treated as in Fig. 2. The females were mated for 48 h. A: percentage of mated females that had pups. B: average number of pups per mother that had pups. C: pup body weight at PND13. *P < 0.05, compared with corresponding basal diet group; n = 4 experiments.

Fig. 5.

γ-Tocopherol supplementation increased lung eosinophils in OVA-challenged pups from allergic mothers. Mice were treated as in Fig. 2. A: bronchoalveolar lavage (BAL) leukocytes of PND13 OVA-challenged pups from allergic and saline (nonallergic) control mothers with basal diet or γ-tocopherol-supplemented diet. *P < 0.05 compared with OVA/basal group. **P < 0.05, compared with all groups; n = 8–10 pups/group. B: PND13 OVA-specific serum IgE as determined by ELISA; n = 8–10 mice/group. *P < 0.05, compared with the corresponding basal groups; n = 8–10 pups/group. C: mother OVA-specific serum IgE on gestational day 18 as determined by ELISA; n = 10–15 mothers/group. *P < 0.05, compared with the corresponding basal groups. D: representative micrographs of perivascular regions in pup lung tissue stained with eosin and methyl green. Images were obtained with a ×20 objective on an Olympus Microscope. Arrows on the images indicate some of the eosin-labeled perivascular eosinophils. V, vessel lumen.

Fig. 6.

Maternal supplementation with d-γ-tocopherol increased CCL11, CCL24, and IL-5 in the lung lavage of OVA-challenged pups. BAL supernatants were collected from PND13 OVA-challenged pups from allergic and saline (nonallergic) control mothers with basal diet or γ-tocopherol-supplemented diet. The indicated cytokines and chemokines were analyzed by ELISAs. A: CCL11. B: CCL24. C: IL-5l. *P < 0.05, compared with basal-saline group. **P < 0.05, compared with all saline groups; n = 8–10 mice/group.

Fig. 7.

Maternal supplementation with γ-tocopherol increased mediators in OVA-challenged pup lungs from allergic mothers. Supernatants from 2-h cultures were from 3 OVA-challenged PND13 pup lungs from allergic mothers with γ-tocopherol-supplemented or basal diet. A: supernatants were analyzed with the RayBiotech L-308 array that detects 308 growth factors/cytokines/chemokines. Shown is fold change: the relative expression of the protein from lungs of OVA-challenged pups from γ-tocopherol-supplemented allergic moms divided by the relative expression of the protein for lungs of OVA-challenged pups from allergic moms with basal diet. A value above 1.5 are considered by RayBiotech as candidates for ELISA analysis; n = 6 arrays (3 mice × 2 groups). B: supernatants from the 2-h lung cultures were analyzed for amphiregulin protein by ELISA. C: supernatants from the 2-h lung cultures were analyzed for activin A protein by ELISA. D: supernatants from the 2-h lung cultures were analyzed for GM-CSF protein by ELISA. *P < 0.05, compared with basal-saline group. **P < 0.05, compared with all groups; n = 8–10 mice/group.

Fig. 8.

d-γ-Tocopherol supplementation of allergic female mice increased the numbers of CD11c⁺CD11b⁺ DCs in the pup lung. The lung tissues were from the pups treated as in Fig. 2. Lung DCs were isolated, immunolabeled, and examined by flow cytometry. A: chart of lung CD11c⁺ subsets analyzed in the pup lungs. B: numbers of CD11b⁺ DC subsets per million lung cells. C: numbers of CD11b⁻ DC subsets per million lung cells. Presented is the mean ± SE; n = 8–10 mice/group. *P < 0.05, compared with basal-saline group. **P < 0.05, compared with all other groups.

Fig. 9.

d-γ-Tocopherol supplementation of allergic female mice did not alter the expression of MHCII, CD80, or IRF4 on the CD11c⁺CD11b⁺ pup lung DCs. The pup lung cells were those analyzed in Fig. 8. Presented is the mean fluorescence intensity (MFI) for MHCII, CD80, or IRF4. A: MFI of MHCII on mDCs. B: MFI of MHCII on alveolar DCs. C: MFI of MHCII on resident DCs. D: MFI of CD80 on mDCs. E: MFI of CD80 on alveolar DCs. F: MFI of CD80 on resident DCs. G: MFI of IRF4 in mDCs. H: MFI of IRF4 in alveolar DCs. I: MFI of IRF4 on resident DCs. Presented is the mean ± SE; n = 8−10 mice/group.

Fig. 10.

d-γ-Tocopherol supplementation of allergic female mice increased the numbers of CD11c⁺CD11b⁺ DCs in the fetal liver. The mice were treated as in Fig. 2 and fetal livers were collected on GD18. The liver cells were isolated, immunolabeled and examined by flow cytometry. A: chart of lung CD11c⁺ subsets analyzed in the GD18 fetal liver. B: numbers of CD45⁺CD11c⁺CD11b-Ly6c⁺CD103-PDCA⁺MHCII⁺ plasmacytoid DC phenotype in the fetal liver. *P < 0.05, compared with corresponding mothers with basal diet. Ca-Cc: numbers of mDC subsets per million fetal liver cells. Cd-Cf: MFI of MHCII, CD80 and IRF4 on fetal liver cells with the mDC phenotype. Dg-Di: numbers of resident DC subsets per million fetal liver cells. Dj-Dl: MFI of MHCII, CD80, and IRF4 on fetal liver cells with the resident DC phenotype. Presented is the mean ± SE. *P < 0.05, compared with basal groups. **P < 0.05 compared with all other groups; n = 8–10 mice/group.

Fig. 11.

d-γ-Tocopherol increased the generation of bone marrow-derived IRF4⁺CD11c⁺CD11b⁺ DCs in vitro. Bone marrow from PND10 pups with basal diet was cultured for 10 days with GM-CSF in the presence of 0.01% DMSO (solvent control) or 2 μM d-γ-tocopherol (as we previously described for in vitro cell loading with d-α-tocopherol) (7). The cells were nonstimulated or stimulated with 20 μg/ml house dust mite (HDM) extract overnight. The DCs were analyzed by immunolabeling and flow cytometry. Presented is the number of IRF4⁺CD45⁺CD11b⁺CD11c⁺ MHCII⁺ DCs. There was no difference in the %live cells between the 2 groups (data not shown). There were no cells from the culture with the monocyte-derived phenotype (CD45⁺CD11c⁺CD11b⁺Ly6c⁺, MHCII high; data not shown); n = 5–6 from a representative experiment of 2 experiments. Presented is the mean ± SE. *P < 0.05, compared with all groups. **P < 0.05, compared with nonstimulated-DMSO group. #P < 0.05, compared with nonstimulated-DMSO control group and compared with HDM-stimulated γ-tocopherol-treated group.