Endothelial stroma programs hematopoietic stem cells to differentiate into regulatory dendritic cells through IL-10

Abstract

Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11b hi Ia lo DCs secreting high levels of TGF-␤, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. IntroductionDendritic cells (DCs) play crucial roles in the initiation and regulation of immune responses. 1,2 The ability of DCs to initiate immune responses or induce tolerance is strictly dependent on their maturation state or subsets. It has been reported that immature DCs that are deficient of costimulatory molecules can induce T-cell anergy, generate regulatory T (Treg) cells, and promote alloantigen-specific tolerance. Several types of DCs with negative regulatory functions have been reported. 3 Most regulatory DCs are prepared in vitro using immunosuppressive cytokines, such as IL-10 and TGF-␤. [4][5][6] However, this may not reflect the real differentiation of regulatory DCs in the immune microenvironment in vivo.The development of hematopoietic cells in vivo occurs in the context of microenvironment or niche, 7 which consists of many types of stromal cells, such as fibroblasts, macrophages, endothelium cells, and adipose cells. The microenvironment provides various signals for hematopoietic cell development. 7 Different microenvironments support different types of cell differentiation. The spleen is an important lymphoid organ, and the mouse spleen maintains hematopoietic function throughout life. 8 Furthermore, hematopoietic stem cells (HSCs) are found in spleens of adult mice. 8 Therefore, it is conceivable that HSCs in the spleen may differentiate into different immune cells in situ.Splenic stromal cells cultured in vitro could mimic the splenic microenvironment in vivo to some extent, despite their differences in some constituents. There is evidence that long-term cultured splenic stromal cells can support the development of dendritic-like cells in the absence of exogenous cytokines, and the dendritic-like cells have the phenotype and function of DCs, 9-13 strongly suggesting that splenic microenvironment could induce hematopoietic progenitors to differentiate directly into DCs. Stromal cells cultured in vitro consist of multiple components. Purification of the various components will help to study the role of specific cell type in the induction of DCs. We established the method of preparing endothelial splenic stroma cells (ESSCs) and i...

Pulmonary stromal cells induce the generation of regulatory DC attenuating T‐cell‐mediated lung inflammation

“…Splenic DCreg inhibit T-cell proliferation via NO, whereas pulmonary DCreg inhibit T-cell proliferation via PGE2. We also have data showing the difference between lung and spleen microenvironment in regulating DC differentiation; that is, splenic stromal cells could program hematopoietic stem cells to differentiate into DCreg [8], whereas MPSC failed to do so (data not shown).…”

“…Before collection by FACSCalibur (Becton Dickinson, Mountain View, CA), 1 Â 10 5 3-mm PE-labeled beads were added to each well as an internal control. Finally, cells and beads were collected at high speed for 52 s. Total viable CD4 1 cells in each well were calculated according to the formula: number total 5 (number viable CD4 /number beads ) Â 10 5 .Assays for antigen-specific T-cell response Although DO11.10 T cells are the responder T cells specific for chicken OVA (cOVA)/I-Ad (BALB/c), it has been proven that DO11.10 T cells can also cross-react with cOVA/I-Ab (C57BL/6) [35,36], and our previous data [7,8] demonstrate that C57BL/6 DC can present OVA antigen to CD4 T cells from DO11.10 Â C57BL/6 F1 hybrid mice and cause CD4 T-cell activation and proliferation. Thus, to determine the antigenpresenting ability of different DC, splenic CD4 T cells from DO11.10 Â C57BL/6 F1 hybrid mice positively selected by MACS were co-cultured with viable DC in the presence of OVA [323][324][325][326][327][328][329][330][331][332][333][334][335][336][337][338][339] peptide at a ratio of 1:10 (DC:T) in round-bottom 96-well plates for 5 days.…”

“…Up to now, regulatory DCreg were generated by culturing imDC in the presence of immunosuppressive cytokines such as IL-10 and TGF-b or immunomodulatory drugs [4][5][6], which does not represent the in vivo physiologic conditions of DCreg in the organ microenvironment. More and more data show that the microenvironment in certain tissues has the ability to induce DC development [7][8][9][10] and also affects the function of DC; for example, DC in brain and kidney display regulatory functions but not T-cell-activating functions [11,12]. Our previous studies demonstrate that endothelial splenic stromal cells, which are used to mimic the in vivo splenic microenvironment, can promote the proliferation of mature DC (maDC) [7] and hematopoietic stem cells [8], driving them to à These authors contributed equally to this work.…”

“…More and more data show that the microenvironment in certain tissues has the ability to induce DC development [7][8][9][10] and also affects the function of DC; for example, DC in brain and kidney display regulatory functions but not T-cell-activating functions [11,12]. Our previous studies demonstrate that endothelial splenic stromal cells, which are used to mimic the in vivo splenic microenvironment, can promote the proliferation of mature DC (maDC) [7] and hematopoietic stem cells [8], driving them to à These authors contributed equally to this work. The lung is one of the organs connected with the outside environment of the organism and interfaces a vast quantity of environmental antigens, some of which are dangerous but some are innocent.…”

“…Splenic DCreg inhibit T-cell proliferation via NO, whereas pulmonary DCreg inhibit T-cell proliferation via PGE2. We also have data showing the difference between lung and spleen microenvironment in regulating DC differentiation; that is, splenic stromal cells could program hematopoietic stem cells to differentiate into DCreg [8], whereas MPSC failed to do so (data not shown).Regarding the mechanisms of pulmonary stroma-driven differentiation of imDC to DCreg, we have excluded the involvement of VEGF, IL-6, M-CSF and PGE2 and finally confirmed that pulmonary stroma-derived TGF-b is involved in the differentiation of DCreg from imDC. As reported before, TGF-b can down-regulate the antigen-presenting function of DC and also can be used to induce DCreg ex vivo.…”