Mouse Splenic Dendritic Cells Research Articles

On the size-regulation of RNA-loaded lipid nanoparticles synthesized by microfluidic device.

The use of lipid nanoparticles (LNPs) for nucleic acid delivery is now becoming a promising strategy with a number of clinical trials as vaccines or as novel therapies against a variety of genetic and infectious diseases. The use of microfluidics for the synthesis of the LNPs has attracted interest because of its considerable advantages over other conventional synthetic methods including scalability, reproducibility, and speed. However, despite the potential usefulness of large particles for nucleic acid delivery to dendritic cells (DCs) as a vaccine, the particle size of the LNPs prepared using microfluidics is typically limited to approximately from 30 to 100nm. In this study, focusing on Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the effect of some synthetic parameters, including total flow rate, flow rate ratio, buffer pH, lipid concentration, molar ratio of PEG-lipid as well as salt concentration, on particle size was systematically examined by means of the design of experiment approaches. The findings indicated that the simple addition of salt (e.g. NaCl) to a buffer containing nucleic acids contributed greatly to the synthesis of large LNPs over 200nm and this effect was concentration-dependent with respect to the salt. The effect of salt on particle size was consistent with a Hofmeister series. The systemic injection of larger mRNA-loaded LNPs resulted in a higher transgene expression in mouse splenic DCs, a higher activation of various splenic immune cells, and had a superior effect as a therapeutic cancer vaccine in a syngeneic mouse model compared to the smaller-sized counterpart with constant lipid composition prepared with lower NaCl concentration. Collectively, size-regulation by the simple addition of salt is a promising strategy for developing potent LNPs.

Dual Roles of PU.1 in the Expression of PD-L2: Direct Transactivation with IRF4 and Indirect Epigenetic Regulation

PD-L2, which has been identified as a PD-1 ligand, is specifically expressed in dendritic cells (DCs) and macrophages. The transcription factors that determine the cell type-specific expression of PD-L2 are largely unknown, although PD-1 and its ligands, which have been shown to play important roles in T cell suppression, have been vigorously analyzed in the field of cancer immunology. To reveal the mechanism by which Pdcd1lg2 gene expression is regulated, we focused on DCs, which play key roles in innate and acquired immunity. The knockdown of the hematopoietic cell-specific transcription factors PU.1 and IRF4 decreased PD-L2 expression in GM-CSF-induced mouse bone marrow-derived DCs. Chromatin immunoprecipitation assays, luciferase assays, and electrophoretic mobility shift assays demonstrated that PU.1 and IRF4 bound directly to the Pdcd1lg2 gene via an Ets-IRF composite element sequence and coordinately transactivated the Pdcd1lg2 gene. Furthermore, PU.1 knockdown reduced the histone acetylation of the Pdcd1lg2 gene. The knockdown of the typical histone acetyltransferase p300, which has been reported to interact with PU.1, decreased the expression and H3K27 acetylation of the Pdcd1lg2 gene. GM-CSF stimulation upregulated the Pdcd1lg2 gene expression, which was accompanied by an increase in PU.1 binding and histone acetylation in Flt3L-generated mouse bone marrow-derived DCs. The involvement of PU.1, IRF4, and p300 were also observed in mouse splenic DCs. Overall, these results indicate that PU.1 positively regulates Pdcd1lg2 gene expression as a transactivator and an epigenetic regulator in DCs.

Abstract 625: Modest Elevations of Extracellular Sodium Activate Dendritic Cells and Contribute to Hypertension

Recently, it has become apparent that sodium can accumulate in the interstitium of hypertensive humans and animals, and such high salt concentrations drive immune cells toward a pro-inflammatory phenotype through poorly defined mechanisms. We hypothesized that dendritic cells (DCs) are activated by modest elevations of extracellular sodium via formation of IsoLGs leading to hypertension. Isolated mouse splenic DCs were cultured in media with either normal salt (NS, 150 mM NaCl), high salt (HS, 190 mM NaCl) or HS plus the gp91ds-tat peptide which inhibits assembly of NADPH oxidase for 24 hours. DCs exposed to HS demonstrated a marked increase in superoxide production compared to NS (146.3±9.5 vs. 100.0±5.0 % control, p<0.001). This was NADPH oxidase dependent because incubation with the gp91ds-tat peptide prevented this effect. Using flow cytometry, we found that DCs cultured in HS had a significantly higher expression of CD86 and more IsoLG-protein adducts than those cultured in NS. These effects were not mimicked by an equiosmolar concentration of mannitol. Western blot analysis of protein extracts from DCs indicated HS markedly increased phosphorylation of the NADPH oxidase subunit p47 phox and association of p47 phox with gp91 phox . Importantly, we found that the salt-activated DCs promote production of cytokines interferon gamma and interleukin 17 by primed T cells. To determine whether these salt-activated DCs promote hypertension, mice received adoptive transfer of splenic DCs that were cultured for 24 hours in either NS (n = 6); HS (n = 5); or HS plus an IsoLG scavenger (n = 6). Mice were then implanted with radiotelemeters to measure mean arterial pressure (MAP) and infused with a subpressor dose (140 ng/kg/min) of angiotensin II (AngII) for two weeks. This caused no increase in MAP in mice that received NS DCs (+0.8 +/- 3.3 mmHg), whereas MAP increased significantly with AngII infusion in mice that received HS DCs (+13.4 +/- 5 mmHg). This pro-hypertensive effect of salt on DCs was attenuated by scavenging of IsoLGs (-3.3 +/- 4.4 mmHg) during salt exposure. These findings provide a mechanistic link between salt, inflammation and hypertension involving increased oxidative stress and IsoLG production in DCs.

SH2 domain-containing adaptor protein B expressed in dendritic cells is involved in T-cell homeostasis by regulating dendritic cell-mediated Th2 immunity.

PurposeThe Src homology 2 domain–containing adaptor protein B (SHB) is widely expressed in immune cells and acts as an important regulator for hematopoietic cell function. SHB silencing induces Th2 immunity in mice. SHB is also involved in T-cell homeostasis in vivo. However, SHB has not yet been studied and addressed in association with dendritic cells (DCs).Materials and MethodsThe effects of SHB expression on the immunogenicity of DCs were assessed by Shb gene silencing in mouse bone marrow–derived DCs (BMDCs). After silencing, surface phenotype, cytokine expression profile, and T-cell stimulation capacity of BMDCs were examined. We investigated the signaling pathways involved in SHB expression during BMDC development. We also examined the immunogenicity of SHB-knockdown (SHBKD) BMDCs in a mouse atopic dermatitis model.ResultsSHB was steadily expressed in mouse splenic DCs and in in vitro–generated BMDCs in both immature and mature stages. SHB expression was contingent on activation of the mitogen- activated protein kinase/Foxa2 signaling pathway during DC development. SHBKD increased the expression of MHC class II and costimulatory molecules without affecting the cytokine expression of BMDCs. When co-cultured with T cells, SHBKD in BMDCs significantly induced CD4+ T-cell proliferation and the expression of Th2 cytokines, while the regulatory T cell (Treg) population was downregulated. In mouse atopic dermatitis model, mice inoculated with SHBKD DCs developed more severe symptoms of atopic dermatitis compared with mice injected with control DCs.ConclusionSHB expression in DCs plays an important role in T-cell homeostasis in vivo by regulating DC-mediated Th2 polarization.

High Salt Activates Dendritic Cells to Promote Hypertension

Both inflammation and dietary salt intake have been implicated in the pathogenesis of hypertension. We have previously shown that the formation of immunogenic isoketal‐protein adducts in dendritic cells (DCs) plays an important role in the development of experimental hypertension. Recently, it has been shown that salt can accumulate in the interstitial space and promote inflammation. In the current study, we tested the hypothesis that exposure to high salt drives DCs toward an activated state, leading to the production of immunogenic isoketals and the promotion of hypertension. To test this hypothesis, mouse splenic DCs were cultured in media with either normal salt (NS, 150 mM NaCl) or high salt (HS, 190 mM NaCl) for 24 hours. Exposure to HS caused a 2‐fold increase in superoxide production in DCs compared to NS. This was NADPH oxidase‐dependent since incubation with the gp91ds‐tat peptide prevented the increase. HS exposure also led to an increase in the activation markers CD80 and CD86, and doubled the number of DCs containing isoketal‐protein adducts. Moreover, DCs exposed to HS but not NS drove proliferation of both CD4+ (5198.2±2398.6 vs. 15.3±7.1 proliferated cells, p<0.01) and CD8+ (25381.6±9495.6 vs. 9.8±4.1 proliferated cells, p<0.01) T cells. None of these effects were mediated by increased osmolality as addition of mannitol (80 mM) to the media had no effect. Western blots of protein extracts from DCs indicated that all NADPH subunits (p47phox, p22phox, gp91phox and p67phox) were increased by exposure of cells to HS, and that these effects were prevented by inhibition of the serum‐and‐glucocorticoid‐inducible kinase‐1 (SGK1). In additional experiments, mice received adoptive transfer of splenic DCs that were cultured for 24 hours in either NS (n = 5); HS (n = 5); HS plus the SGK1 inhibitor, GSK 650394 (HS+GSK, n = 5) or HS plus the isoketal scavenger, 2‐hydroxybenzylamine (HS+2‐HOBA, n=5). Mice were then implanted with radiotelemeters to measure arterial pressure. Following recovery and two days of baseline, subcutaneous osmotic minipumps were implanted for administration of a generally sub‐pressor dose of angiotensin II (140 ng/kg/min). This caused no increase in blood pressure in mice that received NS DCs, whereas mean arterial pressure increased significantly (14 ± 4 mmHg, p<0.05) by one week of angiotensinogen II infusion in mice that received salt‐activated DCs. The pro‐hypertensive effect of salt on DCs was completely blocked by inhibition of SGK1 and scavenging of isoketals during their salt exposure. Taken together, our data indicate that DCs can be activated by exposure to a high salt environment which can exist in pro‐hypertensive states, and that this likely involves increased NADPH oxidase mediated superoxide production and formation of isoketal‐protein adducts. Moreover, high salt exposure can cause DCs to become pro‐hypertensive. These studies define a new pathway linking salt to immune activation and identify a previously undefined role of SGK1 in this process.Support or Funding InformationThis work is supported by National Institutes of Health grants R01HL039006, P01HL058000, P01HL095070, P01GM015431, R01HL108701, R01HL105294, VITA award HHSN268201400010C and the Strategically Focused Research Network Award from the American Heart Association.

Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation.

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.

Abstract 105: High Salt Activates Immune Cells to Promote Hypertension

High salt intake and inflammation are implicated in the genesis of hypertension. We have previously shown that hypertension activates dendritic cells (DCs), by promoting the formation of immunogenic isoketal-protein adducts. Recently it has become clear that sodium can accumulate in the interstitial space and promote inflammation. We hypothesized that high salt activates antigen presenting cells including monocytes and DCs to produce immunogenic isoketals. Exposure of mouse splenic DCs to high salt (190 mM) for 24 hours led to an increase in superoxide production compared to regular RPMI media (146.3±9.5 vs. 100.0±5.0 % control, p<0.001). This was NADPH oxidase dependent because incubation with the gp91ds-tat peptide prevented this effect. High salt exposure also led to an increase in the activation markers CD80, CD86, and a 48% increase in DCs containing isoketal-protein adducts. Moreover, DCs exposed to high salt drove T cell proliferation (5198.2±2398.6 vs. 15.3±7.1 proliferated CD4+ cells and 25381.6±9495.6 vs. 9.8±4.1 proliferated CD8+ cells, p<0.05). This was not due to increased osmolality, as mannitol did not mimic these effects. Western blots of protein extracts from DCs indicated that all NADPH subunits (p47phox, p22phox, gp91phox and p67phox) were increased by exposure of cells to high salt, and that these effects were prevented by inhibition of the salt-sensing glucokinase (SGK1). In additional studies, we found that human monocytes, which are precursors of myeloid DCs, from hypertensive subjects possess higher CD86 and isoketal-protein adducts compared to normotensives, and that these are further increased by exposure to 190 mM salt for 48 hours. Taken together, our data indicate that antigen presenting cells are activated by exposure to the high sodium environment that can occur in pro-hypertensive states, and that this likely involves increased expression and activation of the NADPH oxidase. Our data also describe a previously undefined role of the SGK1 in this process.

Dab2, a negative regulator of DC immunogenicity, is an attractive molecular target for DC-based immunotherapy

Dab2 is an adapter protein involved in receptor-mediated signaling, endocytosis, cell adhesion, hematopoietic cell differentiation, and angiogenesis. It plays a pivotal role in controlling cellular homeostasis. In the immune system, the Dab2 is a Foxp3 target gene and is required for regulatory T (Treg) cell function. Dab2 expression and its biological function in dendritic cells (DCs) have not been described. In this study, we found that Dab2 was significantly induced during the development of mouse bone marrow (BM)-derived DCs (BMDCs) and human monocyte-derived DCs (MoDCs). Even in a steady state, Dab2 was expressed in mouse splenic DCs (spDCs). STAT5 activation, Foxp3 expression, and hnRNPE1 activation mediated by PI3K/Akt signaling were required for Dab2 expression during GM-CSF-derived BMDC development regardless of TGF-β signaling. Dab2-silencing was accompanied by enhanced IL-12 and IL-6 expression, and an improved capacity of DC for antigen uptake, migration and T cell stimulation, which generated strong CTL in vaccinated mice. Vaccination with Dab2-silenced DCs inhibited tumor growth more effectively than did vaccination with wild type DCs. Dab2-overexpression abrogated the efficacy of the DC vaccine in DC-based tumor immunotherapy. These data strongly suggest that Dab2 might be an intrinsic negative regulator of the immunogenicity of DCs, thus might be an attractive molecular target to improve DC vaccine efficacy.

Abstract 2880: Novel alphavirus-based vaccine targets dendritic cells and efficiently breaks immunological tolerance to “self” tumor-associated antigen (PSA) in an HLA-DR mouse model of prostate cancer

Abstract The development of immunotherapy for prostate cancer based on the induction of autoimmunity to prostate-specific differentiation antigens is an attractive concept. In this study, we constructed novel virus-like particles vector (VLPV) platform derived from live attenuated human TC-83 IND vaccine against Venezuelan equine encephalitis virus. We tested the ability of VLPV-PSA particles to target mouse CD11c+ dendritic cells (DCs) using in vitro infection assay. Incubation of mouse splenic DCs with VLPV-PSA resulted in robust accumulation of PSA in the cytoplasm of CD11c+ cells as detected by immunofluorescent staining and confocal microscopy. We also tested the immunogenicity and anti-tumor effect of the VLPV-PSA vaccine in a stringent mouse model of prostate cancer in which tolerance to human PSA expressed as a ‘self’ antigen in the prostate was established in a context of the HLA-DR2b chimeric transgene (DR2bxPSA F1 mice). Preventive vaccination with VLPV-PSA induced strong PSA-specific CD8+ T-cell response as detected by IFNγ ELISPOT assay and intracellular cytokine staining. Potent expansion of PSA-specific CD8+ effector T-cells was found in peripheral blood and spleens of vaccinated mice using PSA peptide-loaded MHC class I dextramers. VLPV-PSA vaccination also induced PSA-specific antibody production in immunized mice with predominance of Th1-associated IgG2a sub-isotype. Injection of TRAMP tumor cells expressing PSA into vaccinated mice resulted in elevated infiltration of tumor site with CD8+ T-cells and rapid elimination of PSA-expressing tumor cells during first several weeks after tumor challenge. Tumor survival analysis revealed significant delay of tumor growth in VLPV-PSA vaccinated mice, especially at the early time-points (p=0.044, Gehan-Breslow test). Interestingly, the combination treatment with VLPV-PSA and the blockade of the CTLA-4-mediated checkpoint did not enhance further CD8 T cell response to PSA, and did not show synergistic anti-tumor effect compared to the VLPV-PSA treatment alone. Our data demonstrate that VLPV-PSA are able to infect mouse DCs and induce production of heterologous antigen (PSA) resulting in efficient break of immunological tolerance to PSA and induction of anti-tumor response in a stringent mouse model of prostate cancer. Citation Format: Vladimir Ryabov, Peter Pushko, Irina Tretyakova, Rikka Saito, Richard B. Alexander, Elena N. Klyushnenkova. Novel alphavirus-based vaccine targets dendritic cells and efficiently breaks immunological tolerance to “self” tumor-associated antigen (PSA) in an HLA-DR mouse model of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2880. doi:10.1158/1538-7445.AM2014-2880

The scavenger receptor MARCO modulates TLR-induced responses in dendritic cells.

The scavenger receptor MARCO mediates macrophage recognition and clearance of pathogens and their polyanionic ligands. However, recent studies demonstrate MARCO expression and function in dendritic cells, suggesting MARCO might serve to bridge innate and adaptive immunity. To gain additional insight into the role of MARCO in dendritic cell activation and function, we profiled transcriptomes of mouse splenic dendritic cells obtained from MARCO deficient mice and their wild type counterparts under resting and activating conditions. In silico analysis uncovered major alterations in gene expression in MARCO deficient dendritic cells resulting in dramatic alterations in key dendritic cell-specific pathways and functions. Specifically, changes in CD209, FCGR4 and Complement factors can have major consequences on DC-mediated innate responses. Notably, these perturbations were magnified following activation with the TLR-4 agonist lipopolysaccharide. To validate our in silico data, we challenged DC‘s with various agonists that recognize all mouse TLRs and assessed expression of a set of immune and inflammatory marker genes. This approach identified a differential contribution of MARCO to TLR activation and validated a major role for MARCO in mounting an inflammatory response. Together, our data demonstrate that MARCO differentially affects TLR-induced DC activation and suggest targeting of MARCO could lead to different outcomes that depend on the inflammatory context encountered by DC.

Depressed production of beta-defensins from mouse splenic dendritic cells following thermal injury and its influence on susceptibility to infection.

Beta-defensins (BDs) and dendritic cells (DC) have been described as major effectors on host antimicrobial innate immunities. In the present study, the ability of DC to produce BDs was explored using DC from normal mice and full-thickness (FT)-burned mice. DCs were isolated from spleens of mice, and 1 × 10(6) cells/ml of them were cultured with LPS or SAC. Culture fluids harvested 24 h after cultivation were assayed for BD1 and BD3 and antibacterial activity (colony-counting, Pseudomonas aeruginosa). Also, DCs were tested for BD mRNAs by RT-PCR. Sixty-five percent of the bacterial killing activity was shown by the culture fluids of splenic DC from normal mice, while only 15 % killing activity was shown by the culture fluids of splenic DC from FT-burned mice. X-irradiated NOD SCID IL-2rγ(null) mice inoculated with splenic DC from FT-burned mice showed increased susceptibility to P. aeruginosa infection compared to those from normal mice. Mice splenic DC expressed BD1 mRNA constitutively and expressed BD3 mRNA after stimulation. These BDs were produced by mice splenic DC. As compared with DC from normal mice, DC from FT-burned mice produced decreased amounts of BD1 and BD3 in their culture fluids. These results indicate that (1) DC from spleens of mice have an ability to produce BDs, and (2) the production of BDs by DC is influenced strongly by thermally injured stress. Since FT-burned mice are susceptible to P. aeruginosa infection, BDs produced by DC may play an important role on the host's antibacterial resistance.

Immunostimulatory effects of Leishmania infantum HSP70 recombinant protein on dendritic cells in vitro and in vivo.

Activation of dendritic cells (DCs) has an important role in immunity against Leishmania. We investigated the effect of Leishmania infantum (L. infantum) heat shock protein 70 recombinant protein (rHSP70) as a vaccine on DC maturation and function. BALB/c mouse splenic DCs were isolated and treated with different concentrations of rHSP70. Maturation markers, cytokine production and capability of DCs to proliferate allogeneic T cells were evaluated. Furthermore, this recombinant protein was injected into BALB/c mice, and expression of CD86, CD40 and MHC class II molecules by their splenic DCs were evaluated. rHSP70 significantly increases the production of IL-12p70 by DCs. It had no effect on allogeneic T-cell proliferation in mixed lymphocyte reaction. It increased IFN-γ and decreased IL-4 cytokine level in mixed lymphocyte reaction supernatant. The in vitro study showed that rHSP70 had no significant effect neither on the percentage of CD40(+), CD86(+) and MHC class II(+) DCs nor on the mean fluorescent intensity. However, in vivo results showed that rHSP70 increases the percentage of CD86-, CD40- and MHC class II-expressing cells as well as mean fluorescent intensity of CD40 and MHC class II. This study demonstrated the capability of L. infantum-derived rHSP70 in maturating BALB/c mice splenic DCs and in vivo polarization of immunity to a Th1 response.

Egr2 induced during DC development acts as an intrinsic negative regulator of DC immunogenicity

Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T-cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC-mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2-knockdown (Egr2-KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co-stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2-KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL-12 expression and Th1 immunogenicity of a DC vaccine. DC-mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2-KD, and impaired by Egr2 overexpression in antigen-pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development.

Modulation of Myeloid-Derived Dendritic Cell Maturity: Unmasking a Novel Role for the Tumor Suppressor p15Ink4b in Immunity

Abstract 2469p15Ink4b, an inhibitor of cyclin-dependent kinases, is a tumor suppressor frequently associated with hematological malignancies. Its inactivation through DNA methylation is one of the most prevalent epigenetic alterations reported in up to 80% of all acute myeloid leukemia (AML) patients. p15Ink4b is also silenced in 50% of patients diagnosed with myelodysplastic syndromes and its silencing correlates with frequent disease progression into AML. During the leukemogenesis process, escape of pre-leukemic cells from immune clearance represents an important step in the establishment of leukemic disease. Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the regulation of immune responses. In immune surveillance, their primary function is to stimulate naïve T cells against pathogens and cancerous cells leading to their effective clearance. However, whether p15Ink4b plays a role in DC development has never been addressed.In this study, we found that expression of p15Ink4b is strongly induced in mouse splenic DCs and during the development of bone marrow-derived DCs (BM-DCs). Increased expression levels were also found during the development of human CD34-derived DCs suggesting an important role for p15Ink4b in DC maturation. To investigate the function of p15Ink4b during the differentiation and maturation of DCs we used the previously generated p15Ink4bfl/fl-LysMcre conditional knockout mice, where a myeloid-specific deletion of p15Ink4b closely mimics inactivation of the gene in AML. The knockout mice developed nonreactive monocytosis and were predisposed to retrovirus-induced AML. These results provided strong experimental evidence for a role of the gene as a tumor suppressor for myeloid leukemia.Myeloid-specific deletion of p15Ink4b in mice resulted in a reduction in the common DC progenitor pool as compared to wild type mice. p15Ink4bfl/fl-LysMcre mice had significantly fewer and less mature myeloid DCs (mDCs) than the wild type mice whereas other DC subtypes were not affected. Consistent with this data, BM cells from the p15Ink4bfl/fl-LysMcre mice cultured in vitro, generated BM-DCs that express lower levels of the antigen presenting (MHCII) and the co-stimulatory (CD80, CD86) molecules when activated with LPS. Re-expression of p15Ink4b in knockout BM-DCs resulted in an increase in the expression of both co-stimulatory molecules confirming a role for p15Ink4b in the regulation of the maturation process of DCs. The incomplete maturation of BM-DCs correlated with a reduced ability to activate T cells in a MHCII-mismatched mixed leukocyte reaction, and to uptake antigen suggesting that loss of p15Ink4b affects the function of BM-DCs.Taken together, our results indicate a novel role for p15Ink4b in mDC development, and suggest that frequent inactivation of p15Ink4b in myeloid malignancies could lead to an inefficient anti-leukemic immune response during leukemogenesis. Our data also have an important translational significance. AML blasts isolated from patients and differentiated ex-vivo into DCs represent a powerful immunotherapy tool. However, AML-DCs have reportedly a partially impaired maturation process as compared to DCs from healthy donors. We propose that re-expression of p15 in AML-DCs may overcome some of the limitations of a DC-based immunotherapy for AML patients. Disclosures:No relevant conflicts of interest to declare.

Pro-inflammatory Cytokine Expression of Spleen Dendritic Cells in Mouse Toxoplasmosis

Dendritic cells have been known as a member of strong innate immune cells against infectious organelles. In this study, we evaluated the cytokine expression of splenic dendritic cells in chronic mouse toxoplasmosis by tissue cyst-forming Me49 strain and demonstrated the distribution of lymphoid dendritic cells by fluorescence-activated cell sorter (FACS). Pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-6, and IL-10 increased rapidly at week 1 post-infection (PI) and peaked at week 3 PI. Serum IL-10 level followed the similar patterns. FACS analysis showed that the number of CD8α+/CD11c+ splenic dendritic cells increased at week 1 and peaked at week 3 PI. In conclusion, mouse splenic dendritic cells showed early and rapid cytokine changes and may have important protective roles in early phases of murine toxoplasmosis.

Expression and function of histamine H4 receptor in mouse splenic dendritic cells

Dendritic cells (DC) are the master regulators of the immune system. DCs act as ‘‘sentinels’’ and they are the most potent antigen-presenting cells specialized for capture, transport, processing and presentation of antigens [1]. During these activities numerous factors may influence their functions. Histamine is one of these mediators released from neighboring mast cells, basophils, and even from the DCs themselves [2]. Histamine exerts its physiological functions through four different G protein-linked receptors: H1R, H2R, H3R, and the recently discovered H4R [3]. In contrast to other histamine receptors, H4R expression shows tissue specificity, it is mainly found in immune cells, e.g. mast cells, eosinophils, T cells, and DCs. Very recently, it has also been detected in the mouse central nervous system [4]. H4R expression has been investigated mainly on the mRNA level, and it has been also described in murine bone marrow-derived DCs. The involvement of H4R in the regulation of DC chemotaxis and cytokine production has been newly published [5]. We have studied the expression of H4R protein in mouse splenic DCs and its possible role in a major DC function, namely antigen presentation.

Porin-incorporated liposome induces Toll-like receptors 2- and 6-dependent maturation and type 1 response of dendritic cell

Porin of Shigella dysenteriae was incorporated in liposome (PIL) and presented to mouse splenic dendritic cells (DC). PIL up-regulated Toll-like receptor (TLR) 2 and TLR6 on DC, showing that co-expression of the two TLRs is involved in recognition of porin. Detection of myeloid differentiating factor 88 (MyD88)-TLR2 complex confirmed interaction between the two for triggering the downstream signaling, which ultimately led to TLR2-dependent nuclear translocation of nuclear factor-kappa B. PIL-induced expression of MHC class II (I-Ab), CD40 and CD80 showed maturation of DC, whereas up-regulation of intercellular adhesion molecule-1 and CCR7 implicated the capacity of splenic DC to migrate. Induction of messenger ribonucleic acid for the chemokines, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta and regulated upon activation, normal T cell expressed and secreted indicated a strong bias of PIL for type 1 polarization that was supported by the intracellular expression and release of tumor necrosis factor (TNF)-alpha and IL-12. Along with CD40 and CD80 expression, release of the cytokines of CD11c+ JAWS II cells was inhibited by TLR2 or simultaneous TLR2 and 6 knockdown showing that recognition of PIL by the two TLRs is essential for DC activation and type 1 polarization. The signaling pathway initiated upon recognition of PIL by the TLRs was MyD88 dependent as confirmed by inhibition of IL-6, TNF-alpha and IL-12 release of MyD88-knockdown JAWS II cells. The maturation and polarization of DC induced T(h)1 phenotype, as evident from proliferation, activation and IFN-gamma release of allogeneic CD4+ T cells in response to PIL-stimulated DC, thereby suggesting that the adjuvant activity of PIL can successfully bridge the innate and adaptive immunity.

Effects of a hexameric deoxyriboguanosine run conjugation into CpG oligodeoxynucleotides on their immunostimulatory potentials.

CpG oligodeoxynucleotides (ODNs) are promising immunomodulatory agents for treating human diseases and vaccine development. Phosphodiester CpG ODNs were demonstrated to have poor immunostimulatory potentials for cytokine production. However, the conjugation of consecutive deoxyriboguanosine residues, called a dG run, at the 3' terminus of phosphodiester CpG ODNs significantly enhanced TNF-alpha and IL-12 production from mouse splenic dendritic cells (DCs). The optimal induction of cytokine production was achieved by the addition of a hexameric dG (dG6) run. In contrast, the existence of a dG6 run either at the 5' terminus of phosphodiester CpG ODNs or at the 3' terminus of phosphorothioate CpG ODNs diminished CpG-mediated cytokine induction, suggesting that the effects of a dG run depend on its location and the chemical property of the ODN backbone, respectively. In addition, we provided the evidence that the conjugation of a dG6 run caused the structural transformation of CpG ODNs, which facilitates their targeting into mouse APCs such as splenic DCs, B cells, and peritoneal macrophages with a scavenger receptor type A ligand specificity. Among primary APCs, DCs were the most potent for CpG ODN-mediated IL-12 production. Furthermore, we demonstrated that the conjugation of a dG6 run into the 3' terminus of phosphodiester CpG ODNs was crucial for their ability to generate Th1 immunity in vivo. Thus, the conjugation of a dG6 run into phosphodiester CpG ODNs would be an alternative way to optimize their immunostimulatory potentials in vitro and in vivo.

Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells.

Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell-inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell-derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon gamma effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.

Photodynamic alteration of the surface receptor expression pattern of murine splenic dendritic cells.

The photosensitizer benzoporphyrin-derivative monoacid ring A (BPD-MA, verteporfin), in combination with visible light irradiation, a clinical procedure termed photodynamic therapy (PDT), has immunomodulatory activity in various mouse models. We studied the impact of BPD-MA and light upon DBA/2 mouse splenic dendritic cells (DC), a potent antigen-presenting cell (APC) type. DC treated with nanomolar amounts of BPD-MA and 690 nm wavelength light had a reduced capacity to stimulate the proliferation of alloreactive T cells. Treatment with BPD-MA and light reduced DC levels of major histocompatibility (MHC) Class I and II antigens, intercellular adhesion molecule-1 (ICAM-1, CD54), the costimulatory B7-1 (CD80) and B7-2 (CD86) molecules, leucocyte common antigen CD45, the apoptosis-regulating Fas (CD95) receptor and the integrin CD11c. In contrast, DC expression of leucocyte function-associated-1 (LFA-1, CD11a), Mac-1 (CD11b), integrin beta2 chain (CD18) and the DEC-205 receptor increased, while CD40 levels were relatively unchanged 24 h after the treatment. MHC Class I and ICAM-1 levels decreased to 40% of control levels within 2 h following the photodynamic treatment. In the absence of light, BPD-MA did not affect DC receptor levels. Changes in DC receptor levels produced by BPD-MA and red light were similar to those produced by ultraviolet B light irradiation. The photodynamic treatment of activated splenic B cells, a separate APC class, had little effect upon receptor expression, except that MHC Class II levels were moderately decreased 24 h later. Changes in DC receptor expression may contribute to the immunomodulatory action of PDT.