A Novel Function Of Peyer’s Patches Eosinophils:Inducing The Differentiation Of Naive T Cell Into Regulatory T Cell Via Retinoic Acid

Background:Infectious diseases caused by pathogenic microorganism are one of the main threat to human health and life. It is well known that the mucosa is the largest immune organ in the body, and it is generally believed that almost all infectious diseases are initiated at mucosal surface. However, little is known about mucosal immunity against pathogens infection.Within the immune system, a series of anatomically distinct compartments can be distinguished, each of which is specially adapted to generate a response to pathogens present in a particular set of body tissues. The previous part of the chapter illustrated the general principles underlying the initiation of an adaptive immune response in the compartment comprising the peripheral lymph nodes and spleen. This is the compartment that responds to antigens that have entered the tissues or spread into the blood. A second compartment of the adaptive immune system of equal size to this, and located near the surfaces where most pathogens invade, is the mucosal immune system.The gastrointestinal tract is most classic component of the body’s mucosal immune system. In fact, the intestine possesses the largest mass of lymphoid tissue in the human body. The intestinal immune system must constantly maintains immunological tolerance to harmless food antigens and commensal bacteria yet recognizes harmful pathogens and responses to eliminate them. The mechanisms that maintain this balance of intestinal immune homeostasis are poorly understood. Gut-associated lymphatic tissue (GALT) maintain the symbiosis between commensal bacteria and the gut for the fine balance of intestinal immune homeostasis. When the GALT receives signals from the intestinal flora or food antigens, it must limit the magnitude of effector responses and allow the establishment of immunological tolerance. Regulatory T cells (Treg) play an indispensable role in maintaining self tolerance.Although a role for Treg in the maintenance of immune tolerance has been demonstrated in both humans and mice, the origin of these cells is still not completely understood.Tregs play an indispensable role in maintaining self tolerance, aside from Tregs arise in the thymus, peripheral conversion of Tregs occurs primarily in the GALT, suggested that the GALT microenviroment is particularly well suited for peripheral conversion of Treg. It has been proved that retinoic acid promote de novo generation of Foxp3+Treg cells via retinoic acid (RA)The vitamin A metabolite RA is a lipophilic molecule that controls the activity of a constellation of genes via binding to nuclear receptors. Vitamin A is derived from the diet, and the liver constitutes a large reservoir of vitamin A in the form of retinyl esters. Retinyl esters are hydrolyzed to retinol and released into the blood. Once retinol enters cells expressing appropriate enzymes, it is converted successively into retinal and RA. The first step of the conversion is catalyzed by alcohol dehydrogenases and by microsomal retinol dehydrogenases that are expressed by most cells, including dendritic cells (DCs). The second step consists of the oxidation of retinal into RA and is catalyzed by3aldehyde dehydrogenases (ALDHs), known as RALDH1,2, and3and encoded by the Aldhlal,-2, and-3genes, respectively. RALDH expression is limited to certain cell types and, despite the widespread availability of retinol, only cells expressing one of the RALDHs can oxidize retinaldehyde to RA.Recently, DCs that are located in GALT and express Aldhla2have gained considerable attention because of their ability to produce RA. On migration to mesenteric lymph nodes (MLNs), this exclusive property allows them to promote the expression of the gut-tropic α4β7integrin and CCR9chemokine receptor on antigen-responsive T cells and in turn confer them gut-seeking properties. Importantly, RA production by GALT-associated DCs is also involved in the generation of induced Foxp3+Treg(iTreg). Treg can be distinguished from "naturally occurring" Foxp3+Treg (iTreg) on the basis of their development. Whereas nTreg develop in the thymus, iTreg develop de novo in secondary lymphoid organs from conventional, naive CD4+T cells. This conversion that is triggered by DCs requires submitogenic dose of antigen and low costimulation, high levels of transforming growth factor-β (TGF-β) and is greatly enhanced by the presence of RA. The exact mechanism through which DC-produced RA impacts on the generation of iTreg is still a matter of debate. For instance, RA has been proposed to enhance the TGF-β-dependent differentiation of naive CD4+T cells into Foxp3+iTreg by blocking their differentiation into proinflammatory T cells. Alternatively, RA may indirectly affects iTreg generation by preventing memory CD4+T cells from producing cytokines (interleukin-4[IL-4], IL-21, and interferon-y), which inhibit the differentiation of iTreg. RA production by GALT-associated DCs has been proposed to maintain the balance between effector and Treg in the gastrointestinal tract and to constitute a major mechanism underlying oral tolerance. The production of RA by gut DCs is restricted to mDCs expressing the integrin αE chain CD103and requires the presence of both granulocyte-macrophage colony stimulating factor (GM-CSF) and RA in the lamina propria (LP).Considering that, under physiologic conditions, ALDH expression constitutes the only parameter that limits RA production, we used a flowcytometry-based assay to measure ALDH activity at the single-cell level and performed a comprehensive analysis of the RA-producing cell populations present in Peyer’s patches under steady-state conditions.Methods:Isolation of cells with microbeads on LS MACS columns and FACS sorting. To identified the cells with high ALDH activity, we employed flowcytometry-based assay to measure phenotype and May-Grunwald-Giemsa staining to observe morphology. LC (liquid chromatography)/MS/MS assay was used to detect the RA secreting by intestinal eosinophil(Eos), the intestinal cells with high ALDH activity. ELISA assay was performed to test the secretion of TGF-P and IL-2by intestinal Eos. For in vitro stimulation, purified peripheral blood Eos or intestinal Eos were cultured together with unlabeled naive CD4+CD62L+OT-II CD4+T cells, Foxp3expression in CD4+T cells was evaluated using the Foxp3staining set. ELISA assay was performed to test the concentration of IL-4, IL-17, IFN-γ, TGF-β and IL-2in the co-culture supernatant.Results:There is no significant difference among the ALDH activity of three distinct DC in Peyer’s patches. However, a group of cells(CD11c+CD11b+CD8-) in intestinal expressed high level of ALDH activity. CD11c+CD11b+CD8-intestinal cells had moderate expression of Siglec-F+CCR3+, which indicated a Eos character. The image show that the CD11c+CD11b+CD8-intestinal cells are Eos with uniquely shaped nuclei and eosinophilic granules. The RA and TGF-β secreting ability of intestinal eosinophils were demonstrated by culture supernatant quantification. After coculture with naive CD4+CD62L+OT-II CD4+T cells in the presence of antigen and TGF-β, approximately6.5%of cells expressed the marker Foxp3and CD25. After coculture with naive CD4+CD62L+OT-II CD4+T cells in the presence of antigen and TGF-β, approximately6.5%of cells expressed the marker Foxp3and CD25. Culture with intestinal eosinophils lead to strong reduction of IFN-y and IL-17, and correlated with increased TGF-β production. The expression of phenotype is different between intestinal and peripheral blood Eos, which is no expression of CD11c and CD80. Peripheral blood Eos have low level of ALDH activity and no RA secreting. After coculture with naive T cells in the presence of antigen and TGF-β, almost no cells expressed the marker Foxp3and CD25.Conclusion:Intestinal Eos inducing the differentiation of naive T cell into regulatory T cell, and inhibiting the differentiation of Th1and Th17. The mechanism is cause via RA and TGF-β. The expression of phenotype is different between intestinal and peripheral blood Eos. Peripheral blood Eos cannot induce the differentiation of naive T cell into regulatory T cell.