Preliminary Study On The Mechanisms And Functions Of Intestinal Dendritic Cells Subsets In Food Allergies

ObjectiveFood allergy mediated by Cholera toxin (CT), the adjuvant for intestinalmucosal immunity, was investigated in our experiment. The contribution ofintestinal dendritic cells (DC) subsets to CT–induced food allergy by breakingtolerance is the key scientific tissue, and the modern cell/molecularimmunologic methods are used as the core technology. By combined applicationof transgenic and knockout mice, this experiment studied the effects of CT onthe intestinal DC subsets in innate immunity, as well as regulation of theadaptive immune response by intestinal DC subsets upon CT stimulation. We tryto understand the effect and possible mechanism of the intestinal DC subsetsinvolved in CT-induced food allergy. This study helps to provide the theoreticalbasis for developing drugs and methods of prevention and treatment of foodallergy in the future.MethodsCD4~+T cells sorted from IL-4-GFP-DO11.10hybrid mouse by magneticbead or KJ126~+CD4~+T cell sorted by flow cytometry were transferred i.v. intoBalb/c mice, followed by oral administration of ovalbumin (OVA) with orwithout CT. A week later, single cell suspension prepared from MLN and PPwere analyzed by flow cytometry to detect IL-4expression on CD4~+T cells,evaluating the induction of Th2response.After mice were administrated by CT,lamina propria (LP), mesenteric lymph node（MLN) and Peyer’s patch (PP) wereisolated for preparation of single cell suspension at day1, day3and day6respectively. Flow cytometry was used to analyze the changes of DC subsets indifferent time points and the expression of the costimulatory molecules in eachDC subsets stained by multicolor immunofluorescence. We also sorted CD4~+T cells from Foxp3-GFP×DO11.10hybrid mouse or CD4~+KJ126~+Foxp3-T cells,and transferred i.v. them into Balb/c mice. Transferred mice were subsequentlygiven OVA with or without CT. A week later, single cell suspension preparedfrom LP, MLN and PP were analyzed by flow cytometry to determine theproliferation of OVA-specific CD4~+T cells and convertion of Foxp3~+CD4~+Tcells from Foxp3-CD4~+T cells, thus finding the type of Treg including naturalTreg(developed from thymus) and induced Treg(differentiated fromFoxP3-cells). Foxp3-GFP mice (C57BL/6background) were orally given OVAwith or without CT, CD4~+T cell proliferation and CD4~+Foxp3~+Treg inductionwere detected by flow cytometry in LP and MLN a week later.Finally, C57BL/6mice and MyD88-/-mice were orally given OVA with or without CT. A weeklater CD4~+Foxp3~+Treg were analyzed in LP, MLN and PP by flow cytometry,investigating the role of Myd88in the development of food allergy induced byCT.ResultsBy transferring CD4~+T cells derived from IL-4-GFP×DO11.10hybridmouse or CD4~+KJ126~+T cells into Balb/c mice, oral administration of OVAcombined with CT promote MLN and PP to produce more IL–4than that of onlyOVA treatment.After CT treatment, the number of migratory DC in MLNsignificantly increase at day1. Expression of CD86, PD-L1and PD-L2onmacrophages in LP, CD103~+CD11b~+DC and CD103~+CD11b-DC subsets in LP,MLN and PP increased at day1after CT treatment. After CD4~+T cells sortedfrom Foxp3-GFP×DO11.10hybrid mouse or purified KJ126~+Foxp3-CD4~+T cellswere transferred i.v. into Balb/c mice, we found that antigen-specific T cellssignificantly increased, the percentage of FoxP3~+Treg in CD4~+T cells decreased,and Foxp3-CD4~+T cells could differentiated into FoxP3~+Treg after oraladministration of OVA and CT. However, oral treatment of only OVA had nosignificant effect. By using Foxp3-GFP mice(C57BL/6background), weobserved that oral administration of OVA combined with CT induced moreCD4~+FoxP3~+Treg in LP, MLN and PP compared with oral administration of onlyOVA. In MyD88-/-mice, oral treatment of OVA with or without CT had nosignificant changes on Foxp3~+CD4~+Treg induction in LP, MLN and PP whencompared with group of oral administration of only OVA. ConclusionOral immunization with CT and OVA induced OVA-specific CD4~+T cellsto differentiation into Th2cells, thus triggering Th2response.CT treatmentleads to an increase of migratory DC in MLN, and upregualtion of CD86, PD-L1and PD-L2on CD103~+CD11b~+DC and CD103-CD11b~+DC in intestinal LP, MLNand PP. By transferring antigen-specific T cells, oral treatment of OVA and CTsignificantly promote CD4~+T cell proliferation in intestinal LP, PP and MLN,while the percentage of Foxp3~+Treg in CD4~+T cells decreased. This resultsuggests that CT treatment can break intestinal immune tolerance by inhibitingTreg induction, though the proliferation of antigen-specific CD4~+T cells isenhanced. Differentiation of transferred antigen-specific KJ126~+Foxp3-CD4~+Tcells into Foxp3~+Treg, proved that CT-induced Treg are iTreg. By usingFoxp3-GFP mice (C57BL/6background), oral administration of CT and OVAlead to a higher percentage of Foxp3~+Treg in CD4~+T cells in LP, MLN and PP,indicating that CT treatment could promote polyclonal CD4~+T cell proliferationand induce Treg.Compared with C57BL/6mice group, there were nosignificantly changes in the percentage of Foxp3~+Treg in CD4~+T cells betweenOVA treatment and OVA~+CT treatment in MyD88-/-mice, indicating thatMyD88-mediated signaling play a key role in the CT-induced food allergy.