The Identification Of CD11^intCD45RB^hiDCreg And A New Subset Of IL-10Produced Macrophages In Vivo And In Vitro

Objective To identify the characteristics, functions, and mechanisms of regulatorydendritic cells which were induced in vivo by intraperitoneal injection of LPS, and comparewith conventional mature dendritic cells in vivo and in vitro. Besides, to identify thecharacteristics and functions of the differential cells which were induced by co-culturingspleen stroma cells with bone marrow mononuclear cells in vitro. Methods The regulartorydendritic cells were identified in vitro by co-cultured with homologous CD4~+CD25-T cellsand innate immunity cells, respectively. First, we set out to analyze whether the regulatorydendritic cells could affect T cell proliferation, apoptosis, and differentiation. Then thepossible underlying mechanisms were investigated through comparing the expression ofIL-10, Arginase-1, iNOS, ROS, PD-L1, PD-L2, and FasL in regulatory dendritic cells andmature dendritic cells. Functional blockade of the overexpressed molecule(s) was employedto see whether the effects of the regulatory dendritic cells could be reversed. Finally, weco-cultured the two subsets of dendritic cells with macrophages induced in vivo and in vitroto observe the production of cytokines and try to analyze the mechanisms. The regulatorydendritic cells were identified in vivo by investigating whether the regulatory dendritic cellscould play a role in murine inflammatory bowel disease. The model of this colitis wasestablished by intravenous injection of CD4~+CD25-T cells to CB-17SCID mice, andintravenous injecting dendritic cells at day0or providing5%DSS water to C57/B6mice. Thebody weight of each mouse was measured weekly. At week4after transfer, we surveyed thecolon length, histological examination, and cytokines produced by mesenteric lymph nodeand so on. In DSS model, we detected the survival rate. In addition, we identified thedifferentiated cells in vitro which were induced by co-culturing spleen stroma cells with bonemarrow mononuclear cells for14days. We first identified surface markers and themorphology. Then we detected the cytokine production with or without LPS stimulation byELISA. Finally, we analyzed the effects of the differentiated cells on T cell proliferation andsurvival. Results The regulatory dendritic cells slightly inhibit the proliferation of T cells,possess ability to induce the generation of Treg cells, and promote apoptosis of activated Tcells which could be reversed by neutralization antibody against FasL. They could also suppress the production of pro-inflammatory cytokines by macrophages. Furthermore, theypotently prevent colitis induced by CD4~+CD25-T cells or DSS. Co-culture of spleen stromacells with bone marrow mononuclear cells leads to the generation of a novel subset ofmacrophages which express high level of IL-10but low level of IL-12. These macrophagescould inhibit the proliferation of T cells, promote the necrosis of activated CD4~+T cells.Conclusions The regulatory dendritic cells which were induced in vivo could negativelyregulate T cells and innate immunity cells. Consequently, they contribute essentially toprevent inflammatory bowel disease. In addition, we have established a method to induce anew subset of macrophages.