Identification of dendritic cell precursor from the CD11c+ cells expressing high levels of MHC class II molecules in the culture of bone marrow with FLT3 ligand.

Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c+MHCII+ or CD11c+MHCIIhi cells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c+ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c+MHCIIhi DC gate were 2A1+ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c+MHCIIhi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c+MHCIIhi cells exhibited a 2A1-CD83-CD115+CX3CR1+ phenotype, and the others consisted of 2A1+CD83+CD115-CX3CR1- and 2A1-CD83-CD115-CX3CR1- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1-CD83-CD115-CX3CR1- cells were immature cDC2s and 2A1+CD83+CD115-CX3CR1- cells were mature cDC2s. Unexpectedly, however, 2A1-CD83-CD115+CX3CR1+ cells, the most abundant cDC2-gated MHCIIhi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCIIhi pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c+MHCIIhiCD115+CX3CR1+ phenotype, in FL-BM culture.

Distinct effects of different adjuvants in the mouse model of allergic airway inflammation.

BACKGROUND: Allergic asthma was typically considered as an inflammatory disease mediated by type 2 immunity. However, recent studies revealed that asthma is a complex disease displaying a variety of phenotypes and endotypes. OBJECTIVE: We examined cellular phenotypes in the mouse model of allergic asthma sensitized with different adjuvants. The aim of our study was to determine immunologic cellular characteristics in mouse asthma models induced by ovalbumin (OVA) and a variety of adjuvants. METHODS: Mice were sensitized intraperitoneally with the admixture of OVA and various adjuvants such as Alhydrogel (alum), papain, lipopolysaccharide (LPS), or CpG, and subsequently challenged with OVA intranasally. The cells in bronchoalveolar lavage (BAL) fluid, lung, and mediastinal lymph node (mLN) were examined by flow cytometric analyses. RESULTS: In the lung and BAL fluid, the highest eosinophil levels were observed in the alum group while the highest neutrophil levels were detected in the LPS group. Meanwhile, the LPS group exhibited the most elevated levels of both RORγt+ innate lymphoid cells (ILCs) and IL-17A+ Th cells in the lung and mediastinal lymph node. In the lung, the number of T-bet+ ILCs was highest in the papain group whereas the number of IFN-γ+ Th cells was highest in the CpG group. CONCLUSIONS: Notable variances are found in the composition of immune cells and expression of cytokines at the site of pathogenesis among the different mouse models of allergic asthma created by the sensitization with different adjuvants.

Global Gene Expression of T Cells Is Differentially Regulated by Peritoneal Dendritic Cell Subsets in an IL-2 Dependent Manner.

Dendritic cells (DCs) in peripheral tissues may have a unique role to regulate innate and adaptive immune responses to antigens that enter the tissues. Peritoneal cavity is the body compartment surrounding various tissues and organs and housing diverse immune cells. Here, we investigated the specialized features of classical DC (cDC) subsets following the intraperitoneal injection of a model antigen ovalbumin (OVA). Peritoneal cDC1s were superior to cDC2s in activating OVA-specific CD8 T cells, while both cDCs were similar in stimulating OVA-specific CD4 T cells. Each peritoneal cDC subset differentially regulated the homing properties of CD8 T cells. CD8 T cells stimulated by cDC1s displayed a higher level of lung-homing receptor CCR4, whereas those stimulated by cDC2s prominently expressed various homing receptors including gut-homing molecules CCR9 and α4ß7. Also, we found that cDC1s played a dominating role over cDC2s in controlling the overall gene expression of CD8 T cells. Soluble factor(s) emanating from CD8 T cells stimulated by peritoneal cDC1s were responsible for mediating this dominance of cDC1s, and we identified IL-2 as a soluble factor regulating the global gene expression of T cells. Collectively, our study indicates that different peritoneal cDC subsets effectively diversify T cell responses by altering the level of cytokines, such as IL-2, in the milieu.

Granulocyte Macrophage-Colony Stimulating Factor Produces a Splenic Subset of Monocyte-Derived Dendritic Cells That Efficiently Polarize T Helper Type 2 Cells in Response to Blood-Borne Antigen.

Dendritic cells (DCs) are key antigen-presenting cells that prime naive T cells and initiate adaptive immunity. Although the genetic deficiency and transgenic overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) signaling were reported to influence the homeostasis of DCs, the in vivo development of DC subsets following injection of GM-CSF has not been analyzed in detail. Among the treatment of mice with different hematopoietic cytokines, only GM-CSF generates a distinct subset of XCR1-33D1- DCs which make up the majority of DCs in the spleen after three daily injections. These GM-CSF-induced DCs (GMiDCs) are distinguished from classical DCs (cDCs) in the spleen by their expression of CD115 and CD301b and by their superior ability to present blood-borne antigen and thus to stimulate CD4+ T cells. Unlike cDCs in the spleen, GMiDCs are exceptionally effective to polarize and expand T helper type 2 (Th2) cells and able to induce allergic sensitization in response to blood-borne antigen. Single-cell RNA sequencing analysis and adoptive cell transfer assay reveal the sequential differentiation of classical monocytes into pre-GMiDCs and GMiDCs. Interestingly, mixed bone marrow chimeric mice of Csf2rb+/+ and Csf2rb-/- demonstrate that the generation of GMiDCs necessitates the cis expression of GM-CSF receptor. Besides the spleen, GMiDCs are generated in the CCR7-independent resident DCs of the LNs and in some peripheral tissues with GM-CSF treatment. Also, small but significant numbers of GMiDCs are generated in the spleen and other tissues during chronic allergic inflammation. Collectively, our present study identifies a splenic subset of CD115hiCD301b+ GMiDCs that possess a strong capacity to promote Th2 polarization and allergic sensitization against blood-borne antigen.

Two Distinct Subsets Are Identified from the Peritoneal Myeloid Mononuclear Cells Expressing both CD11c and CD115.

To this date, the criteria to distinguish peritoneal macrophages and dendritic cells (DCs) are not clear. Here we delineate the subsets of myeloid mononuclear cells in the mouse peritoneal cavity. Considering phenotypical, functional, and ontogenic features, peritoneal myeloid mononuclear cells are divided into 5 subsets: large peritoneal macrophages (LPMs), small peritoneal macrophages (SPMs), DCs, and 2 MHCII+CD11c+CD115+ subpopulations (i.e., MHCII+CD11c+CD115+CD14-CD206- and MHCII+CD11c+CD115+CD14+CD206+). Among them, 2 subsets of competent Ag presenting cells are demonstrated with distinct functional characteristics, one being DCs and the other being MHCII+CD11c+CD115+CD14-CD206- cells. DCs are able to promote fully activated T cells and superior in expanding cytokine producing inflammatory T cells, whereas MHCII+CD11c+CD115+CD14-CD206- cells generate partially activated T cells and possess a greater ability to induce Treg under TGF-ß and retinoic acid conditions. While the development of DCs and MHCII+CD11c+CD115+CD14-CD206- cells are responsive to the treatment of FLT3 ligand and GM-CSF, the number of LPMs, SPMs, and MHCII+CD11c+CD115+CD14+CD206+ cells are only influenced by the injection of GM-CSF. In addition, the analysis of gene expression profiles among MHCII+ peritoneal myeloid mononuclear cells reveals that MHCII+CD11c+CD115+CD14+CD206+ cells share high similarity with SPMs, whereas MHCII+CD11c+CD115+CD14-CD206- cells are related to peritoneal DC2s. Collectively, our study identifies 2 distinct subpopulations of MHCII+CD11c+CD115+ cells, 1) MHCII+CD11c+CD115+CD14-CD206- cells closely related to peritoneal DC2s and 2) MHCII+CD11c+CD115+CD14+CD206+ cells to SPMs.

Viperin Deficiency Promotes Polarization of Macrophages and Secretion of M1 and M2 Cytokines.

Viperin is a multifunctional protein that was first identified in human primary macrophages treated with interferon-γ and in human fibroblasts infected with human cytomegalovirus. This protein plays a role as an anti-viral protein and a regulator of cell signaling pathways or cellular metabolism when induced in a variety of cells such as fibroblasts, hepatocytes and immune cells including T cells and dendritic cells. However, the role of viperin in macrophages is unknown. Here, we show that viperin is basally expressed in murine bone marrow cells including monocytes. Its expression is maintained in bone marrow monocyte-derived macrophages (BMDMs) depending on macrophage colony-stimulating factor (M-CSF) treatment but not on granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. In wild type (WT) and viperin knockout (KO) BMDMs differentiated with M-CSF or G-MCSF, there are little differences at the gene expression levels of M1 and M2 macrophage markers such as inducible nitric oxide synthase (iNOS) and arginase-1, and cytokines such as IL-6 and IL-10, indicating that viperin expression in BMDMs does not affect the basal gene expression of macrophage markers and cytokines. However, when BMDMs are completely polarized, the levels of expression of macrophage markers and secretion of cytokines in viperin KO M1 and M2 macrophages are significantly higher than those in WT M1 and M2 macrophages. The data suggest that viperin plays a role as a regulator in polarization of macrophages and secretion of M1 and M2 cytokines.

Selective SnO x Atomic Layer Deposition Driven by Oxygen Reactants.

SnO x thin films were successfully deposited by the thermal atomic layer deposition (ALD) method using N, N'- tert-butyl-1,1-dimethylethylenediamine stannylene(II) as a precursor and ozone and water as reactants. The growth of SnO and SnO2 films could be easily controlled by employing different reactants and utilizing different ozone and water concentrations, respectively. The formation of both SnO and SnO2 films exhibited typical surface-limiting reaction characteristics, although their growth behaviors differ from one another. The combined studies of density functional theory calculations and experimental analyses showed that the difference in growth behavior of the SnO and SnO2 films can be attributed to the stability of ozone and water on the SnO2 and SnO films. SnO and SnO2 films have different crystal structures and both films were crystallized from the amorphous to polycrystalline states following an increase in the deposition temperature. The absorbance and refractive index of the thin films were investigated using ultraviolet-visible spectroscopy (UV-vis) and spectroscopic ellipsometry (SE), respectively. SnO x films formed using ozone and water as a reactant showed an optical band gap of 3.60-3.17 eV and 2.24-2.30 eV and refractive indices of ∼2.0 and ∼2.6, respectively, which correspond to values typical of SnO2 and SnO. The bilayer structure of SnO/SnO2 was successfully fabricated on indium tin oxide (ITO) glass with nickel as a top electrode at 100 °C. The SnO/SnO2 bilayer exhibited diode characteristics with a current rectification ratio of 15. Our results present a simple but highly versatile growth method for producing multilayer oxide films with electronic properties that can be finely controlled.

Extended Culture of Bone Marrow with Granulocyte Macrophage-Colony Stimulating Factor Generates Immunosuppressive Cells.

Bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow (BM) cells cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for a week. In this study we investigated the effect of duration on the BM culture with GM-CSF. Within several months, the cells in the BM culture gradually expressed homogeneous levels of CD11c and major histocompatibility complex II on surface, and they became unable to stimulate allogeneic naïve T cells in mixed lymphocyte reaction (MLR). In addition, when the BM culture were sustained for 32 wk or longer, the BM cells acquired ability to suppress the proliferation of allogeneic T cells in MLR as well as the response of ovalbumin-specific OT-I transgenic T cells in antigen-dependent manner. We found that, except for programmed death-ligand 1, most cell surface molecules were expressed lower in the BM cells cultured with GM-CSF for the extended duration. These results indicate that BM cells in the extended culture with GM-CSF undergo 2 distinct steps of functional change; first, they lose the immunostimulatory capacity; and next, they gain the immunosuppressive ability.

Skin-Specific CD301b+ Dermal Dendritic Cells Drive IL-17-Mediated Psoriasis-Like Immune Response in Mice.

Conventional dendritic cells (cDCs) are composed of heterogeneous subsets commonly arising from dendritic cell (DC)-committed progenitors. A population of CD301b-expressing DCs has recently been identified in non-lymphoid barrier tissues such as skin. However, whether CD301b+ DCs in the skin represent an ontogenetically unique subpopulation of migratory cDCs has not been fully addressed. Here, we demonstrated that CD301b+ dermal DCs were distinct subpopulation of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent CD11b+ cDC2 lineage, which required an additional GM-CSF cue for the adequate development. Although the majority of lymphoid-resident cDC2 lacked CD301b expression, dermal migratory cDC2 contained a substantial fraction of CD301b+ subset. Similar to CD301b- population, CD301b+ dermal DC development was closely regulated by FLT3 signaling, suggesting their common origin from FLT3L-responsive cDC progenitors. However, FLT3L-driven cDC progenitor culture was not sufficient, but additional GM-CSF treatment was required to produce CD301b+ cDC2. In vivo development of CD301b+ cDC2 was significantly augmented by exogenous GM-CSF, while the repopulation of CD301b+ dermal cDC2 was abrogated by GM-CSF neutralization. Functionally, CD301b+ cDC2 was capable of producing a high level of IL-23, and the depletion of CD301b+ cDC2 effectively prevented IL-17-mediated psoriasiform dermatitis. Therefore, our findings highlight the differentiation program of a distinct CD301b+ dermal cDC2 subset in the skin and its involvement in psoriatic inflammation.

Performance and Stability Enhancement of In-Sn-Zn-O TFTs Using SiO2 Gate Dielectrics Grown by Low Temperature Atomic Layer Deposition.

Silicon dioxide (SiO2) films were synthesized by plasma-enhanced atomic layer deposition (PEALD) using BTBAS [bis(tertiarybutylamino) silane] as the precursor and O2 plasma as the reactant, at a temperature range from 50 to 200 °C. While dielectric constant values larger than 3.7 are obtained at all deposition temperatures, the leakage current levels are drastically reduced to below 10-12 A at temperatures above 150 °C, which are similar to those obtained in thermally oxidized and PECVD grown SiO2. Thin film transistors (TFTs) based on In-Sn-Zn-O (ITZO) semiconductors were fabricated using thermal SiO2, PECVD SiO2, and PEALD SiO2 grown at 150 °C as the gate dielectrics, and superior device performance and stability are observed in the last case. A linear field effect mobility of 68.5 cm2/(V s) and a net threshold voltage shift (ΔVth) of approximately 1.2 V under positive bias stress (PBS) are obtained using the PEALD SiO2 as the gate insulator. The relatively high concentration of hydrogen in the PEALD SiO2 is suggested to induce a high carrier density in the ITZO layer deposited onto it, which results in enhanced charge transport properties. Also, it is most likely that the hydrogen atoms have passivated the electron traps related to interstitial oxygen defects, thus resulting in improved stability under PBS. Although the PECVD SiO2 contains a hydrogen concentration similar to that of PEALD SiO2, its relatively large surface roughness appears to induce scattering effects and the generation of electron traps, which result in inferior device performance and stability.

Competent antigen-presenting cells are generated from the long-term culture of splenocytes with granulocyte-macrophage colony-stimulating factor.

Dendritic cells (DCs) are routinely produced from the culture of mouse bone marrow (BM) with granulocyte-macrophage colony-stimulating factor (GM-CSF) within a period of 10days. Although splenic extramedullary myelopoiesis was suggested to occur under the influence of GM-CSF, the hematopoietic outcome of splenic culture with GM-CSF has not been scrutinized. We have cultured mouse splenocytes with GM-CSF for an extended period of time, where we discovered that the CD11b⁺CD11c⁺ cells began to proliferate prominently after 10days and their number increased until the 4th week of the culture. In parallel experiments, FMS-like tyrosine kinase 3 (FLT3) and its ligand, FLT3L, were not found to influence the culture of splenocytes. Like DCs in the culture of BM with GM-CSF, a distinct population of CD11b⁺CD11c⁺MHC IIhi cells was readily identified as DCs in the long-term culture of splenocytes. After being isolated and plated overnight the CD11b⁺CD11c⁺MHC IIhi cells exhibited non-adherent dendritic morphology, while the other CD11b⁺CD11c⁺ cells became adherent. Besides, these CD11b⁺CD11c⁺MHC IIhi cells possessed relatively weak endocytic and phagocytic abilities but displayed strong antigen-presenting capacities, revealing DC-like characteristics; in contrast, the other CD11b⁺CD11c⁺ cells showed strong endocytosis and phagocytosis of antigens but were poor at antigen presentation, indicating macrophage-like traits. Therefore, we demonstrated that phenotypically as well as functionally genuine DCs are generated in the long-term culture of splenocytes with GM-CSF.

Reduced expression of granule proteins during extended survival of eosinophils in splenocyte culture with GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifaceted hematopoietic cytokine and the culture of mouse bone marrow with GM-CSF produces a variety of myeloid cells including granulocytes, macrophages, and dendritic cells. In the present study, we cultured mouse splenocytes with GM-CSF and examined the changes in hematopoietic cell populations over a week. Most of the splenic hematopoietic cells disappeared significantly from culture within 6days with or without the presence of GM-CSF. Among the splenic granulocyte populations, only eosinophils fully survived throughout the culture with GM-CSF for more than a week. During 10days of culture with GM-CSF, splenic eosinophils maintained their morphology as well as most of their surface molecules at high levels, including CCR3 and Siglec F. Meanwhile, the expression of mRNAs encoding major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO), two major eosinophil-derived granule proteins, was diminished significantly from the cultured eosinophils. EPO assays also revealed that eosinophils in culture for more than 5days retained 30% or less EPO activity compared to those in uncultured splenocytes. In contrast, culture of splenocytes with GM-CSF did not change the capacity of eosinophils to migrate in response to eotaxin-1. Our results indicate that mouse splenic eosinophils are effectively cultured for lengthy periods while their expression of eosinophil-derived granule proteins is specifically suppressed. The relevance of these findings to eosinophilic inflammatory response is discussed.

TCF4-Targeting miR-124 is Differentially Expressed amongst Dendritic Cell Subsets.

Dendritic cells (DCs) are professional antigen-presenting cells that sample their environment and present antigens to naïve T lymphocytes for the subsequent antigen-specific immune responses. DCs exist in a range of distinct subpopulations including plasmacytoid DCs (pDCs) and classical DCs (cDCs), with the latter consisting of the cDC1 and cDC2 lineages. Although the roles of DC-specific transcription factors across the DC subsets have become understood, the posttranscriptional mechanisms that regulate DC development are yet to be elucidated. MicroRNAs (miRNAs) are pivotal posttranscriptional regulators of gene expression in a myriad of biological processes, but their contribution to the immune system is just beginning to surface. In this study, our in-house probe collection was screened to identify miRNAs possibly involved in DC development and function by targeting the transcripts of relevant mouse transcription factors. Examination of DC subsets from the culture of mouse bone marrow with Flt3 ligand identified high expression of miR-124 which was able to target the transcript of TCF4, a transcription factor critical for the development and homeostasis of pDCs. Further expression profiling of mouse DC subsets isolated from in vitro culture as well as via ex vivo purification demonstrated that miR-124 was outstandingly expressed in CD24(+) cDC1 cells compared to in pDCs and CD172α(+) cDC2 cells. These results imply that miR-124 is likely involved in the processes of DC subset development by posttranscriptional regulation of a transcription factor(s).