In humans and mice, offspring of allergic mothers are predisposed to development of allergy. In mice, allergic mothers have elevated β-glucosylceramides (βGlcCers) that are transported to the fetus via the placenta and to offspring via milk. The elevated βGlcCers increase the number of fetal liver CD11c+CD11b+ dendritic cells (DCs) and offspring allergen-induced lung eosinophilia. These effects are modifiable by maternal dietary supplementation with the plant-derived lipids α-tocopherol and γ-tocopherol. It is not known whether βGlcCers and tocopherols directly regulate development of DCs. In this study, we demonstrated that βGlcCers increased development of GM-CSF–stimulated mouse bone marrow–derived DCs (BMDCs) in vitro without altering expression of costimulatory molecules. This increase in BMDC numbers was blocked by α-tocopherol and potentiated by γ-tocopherol. Furthermore, βGlcCers increased protein kinase Cα (PKCα) and PKCδ activation in BMDCs that was blocked by α-tocopherol. In contrast, γ-tocopherol increased BMDC PKCα and PKCδ activation and enhanced the βGlcCer-induced increase in PKCδ activation in a DC subset. Ag processing per DC was minimally enhanced in βGlcCer-treated BMDCs and not altered ex vivo in lung DCs from pups of allergic mothers. Pups of allergic mothers had an increased proportion of CD11b+CD11c+ subsets of DCs, contributing to enhanced stimulation of T cell proliferation ex vivo. Thus, βGlcCer, which is both necessary and sufficient for development of allergic predisposition in offspring of allergic mothers, directly increased development and PKC activation in BMDCs. Furthermore, this was modifiable by dietary tocopherols. This may inform design of future studies for the prevention or intervention in asthma and allergic disease.
Neonatal mice with heterozygous mutations in genes encoding the skin barrier proteins filaggrin and mattrin (flaky tail mice [FT+/−]) exhibit oral peanut-induced anaphylaxis after skin sensitization. As we have previously reported, sensitization in this model is achieved via skin co- exposure to the environmental allergen Alternaria alternata (Alt), peanut extract (PNE), and detergent. However, the function of Alt in initiation of peanut allergy in this model is little understood. The purpose of this study was to investigate candidate cytokines induced by Alt in the skin and determine the role of these cytokines in the development of food allergy, namely oncostatin M (Osm), amphiregulin (Areg), and IL-33. RT-qPCR analyses demonstrated that skin of FT+/− neonates expressed Il33 and Osm following Alt or Alt/PNE but not PNE exposure. By contrast, expression of Areg was induced by either Alt, PNE, or Alt/PNE sensitization in FT+/− neonates. In scRNAseq analyses, Osm, Areg, and Il33 were expressed by several cell types, including a keratinocyte cluster that was expanded in the skin of Alt/PNE-exposed FT+/− pups as compared to Alt/PNE-exposed WT pups. Areg and OSM were required for oral PNE-induced anaphylaxis since anaphylaxis was inhibited by administration of neutralizing anti-Areg or anti-OSM antibodies prior to each skin sensitization with Alt/PNE. It was then determined if intradermal injection of recombinant IL33 (rIL33), rAreg, or rOSM in the skin could substitute for Alt during skin sensitization to PNE. PNE skin sensitization with intradermal rIL33 was sufficient for oral PNE-induced anaphylaxis, whereas skin sensitization with intradermal rAreg or rOSM during skin exposure to PNE was not sufficient for anaphylaxis to oral PNE challenge. Based on these studies a pathway for IL33, Areg and OSM in Alt/PNE sensitized FT+/− skin was defined for IgE induction and anaphylaxis. Alt stimulated two pathways, an IL33 pathway and a pathway involving OSM and Areg. These two pathways acted in concert with PNE to induce food allergy in pups with skin barrier mutations.
In humans and mice, offspring of allergic mothers are predisposed to development of allergy. In mice, allergic mothers have elevated β-glucosylceramides (βGlcCers) that are transported to the fetus via the placenta and to offspring via milk. Elevated βGlcCers increase numbers of fetal liver CD11c+CD11b+ dendritic cells (DCs) and offspring allergen-induced lung eosinophilia. These effects are modifiable by maternal dietary supplementation with the plant-derived lipids α-tocopherol and γ-tocopherol. It is not known whether βGlcCers and tocopherols directly regulate development of DCs. In this study, we demonstrated that βGlcCers (C16:0, C18:0, C18:1, and C24:1) increased development of GM-CSF-stimulated bone marrow-derived DCs (BMDCs) in vitro. This increase in BMDC numbers was blocked by α-tocopherol and potentiated by γ-tocopherol. Furthermore, βGlcCer increased PKCα and PKCδ activation in BMDCs that was blocked by α-tocopherol. In contrast, γ-tocopherol increased BMDC PKCα and PKCδ activation and enhanced the βGlcCer-induced increase in PKCδ activation in a DC subset. Antigen processing per DC was minimally enhanced in βGlcCer-treated BMDCs and not altered ex vivo in lung DCs from pups of allergic mothers. However, lung DCs from pups of allergic mothers had an increased proportion of CD11b+CD11c+ DCs and exhibited enhanced stimulation of T cells ex vivo. Thus, βGlcCer, which is both necessary and sufficient for development of allergic predisposition in offspring of allergic mothers, directly increased development and PKC activation in BMDCs. Furthermore, this was modifiable by dietary tocopherols. This may inform design of future studies for approaches in the prevention or intervention in asthma and allergic disease. Supported by grants from the NIH (U01 AI131337 (Cook-Mills), R01AI127695 (Cook-Mills)) as well as the Pediatric Scientist Development Program (Lajiness) and Marshall Klaus Perinatal Research Award (Lajiness)
Background and Hypothesis: CDllb+CDllc+ dendritic cells (DCs) play a role in the development of allergic disease. It has been shown that of the vitamin E isoforms, α-Tocopherol decreases and γ-Tocopherol increases the generation of bone marrow-derived CD11b+CD11c+ DCs in vivo. And, in vivo experiments have also shown that β-glucosylceramides, endogenous maternal lipids, increase the neonate proliferation of this same subset of DCs. The mechanism for β-glucosylceramide regulation of these specific DC subsets is not known. Furthermore, it is also not known how vitamin E isoforms regulate DC development and differentiation. We determined whether α-tocopherol decreases and γ-tocopherol increases responses to β-glucosylceramide by regulating Protein Kinase C (PKC) activation during CDllb+CDllc+ DC differentiation and proliferation. Project Methods: Cultured bone marrow cells (harvested from mice) were treated with lipid metabolites with and without supplementation of tocopherol isoforms, immunolabeled with antibodies that define DCs and with antibodies that detect active auto phosphorylated forms of PKC. Then, these cells were analyzed using flow cytometry. Results: In vitro β-glucosylceramide elevated DC PKCα/β activity during CDllb+CDllc+ DC differentiation and proliferation/activation. Furthermore, these effects of β-glucosylceramide on DC PKCα/β activity were blocked by α-Tocopherol and elevated by γ-Tocopherol. Potential Impact: These data provides a better understanding of how maternal β-glucosylceramide and dietary supplementation with vitamin E isoforms regulate DC proliferation and differentiation and ultimately development of allergic inflammation in offspring of allergic mothers.
Food allergy can be induced in neonatal mice with heterozygous skin barrier mutations and skin co-exposure to detergent, food allergen and a ubiquitous environmental allergen Alternaria Alternata (Alt) at doses in dust. In this study it was determined whether cytokines are induced by Alt in development of allergy to peanut extract (PNE). In preliminary RNAseq data, Alt induced expression of IL33, oncostatin M (OSM) and amphiregulin (Areg) in the skin of neonatal flakey tail (FT+/−) mice with heterozygous skin barrier mutations. In FT+/−, but not WT neonates, qPCR analyses revealed skin expression of IL33 and OSM following Alt or Alt+PNE exposure compared to neonates with saline skin exposure, while IL33 and OSM were not induced by skin exposure to PNE alone. The expression of Areg was induced by Alt, PNE or Alt+PNE in FT+/− neonates. It was determined whether intradermal injection of recombinant IL33 (rIL33), rAreg or rOSM could substitute for Alt during skin sensitization to PNE. PNE skin sensitization with intradermal rIL33 was sufficient for oral PNE induced anaphylaxis, whereas skin sensitization with intradermal rAreg or rOSM during skin exposure to PNE was not. Injection of neutralizing antibodies for Areg and OSM blocked anaphylaxis in Alt+PNE exposed neonates, indicating that Areg and OSM are required for PNE-induced anaphylaxis prior to skin sensitization. Analysis of skin IL33, Areg and OSM gene expression and plasma protein for IL33, Areg and OSM defined a pathway for IL33, Areg and OSM in Alt+PNE skin. Thus, Alt stimulated expression of IL33 and a pathway of OSM and Areg in concert with PNE to induce food allergy in FT+/− pups. These data have important implications for studies of detection of risks for sensitization to food allergens.
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