Derived Dendritic Cells Function Research Articles

NKL Homeobox Gene VENTX Is Part of a Regulatory Network in Human Conventional Dendritic Cells.

Recently, we documented a hematopoietic NKL-code mapping physiological expression patterns of NKL homeobox genes in human myelopoiesis including monocytes and their derived dendritic cells (DCs). Here, we enlarge this map to include normal NKL homeobox gene expressions in progenitor-derived DCs. Analysis of public gene expression profiling and RNA-seq datasets containing plasmacytoid and conventional dendritic cells (pDC and cDC) demonstrated HHEX activity in both entities while cDCs additionally expressed VENTX. The consequent aim of our study was to examine regulation and function of VENTX in DCs. We compared profiling data of VENTX-positive cDC and monocytes with VENTX-negative pDC and common myeloid progenitor entities and revealed several differentially expressed genes encoding transcription factors and pathway components, representing potential VENTX regulators. Screening of RNA-seq data for 100 leukemia/lymphoma cell lines identified prominent VENTX expression in an acute myelomonocytic leukemia cell line, MUTZ-3 containing inv(3)(q21q26) and t(12;22)(p13;q11) and representing a model for DC differentiation studies. Furthermore, extended gene analyses indicated that MUTZ-3 is associated with the subtype cDC2. In addition to analysis of public chromatin immune-precipitation data, subsequent knockdown experiments and modulations of signaling pathways in MUTZ-3 and control cell lines confirmed identified candidate transcription factors CEBPB, ETV6, EVI1, GATA2, IRF2, MN1, SPIB, and SPI1 and the CSF-, NOTCH-, and TNFa-pathways as VENTX regulators. Live-cell imaging analyses of MUTZ-3 cells treated for VENTX knockdown excluded impacts on apoptosis or induced alteration of differentiation-associated cell morphology. In contrast, target gene analysis performed by expression profiling of knockdown-treated MUTZ-3 cells revealed VENTX-mediated activation of several cDC-specific genes including CSFR1, EGR2, and MIR10A and inhibition of pDC-specific genes like RUNX2. Taken together, we added NKL homeobox gene activities for progenitor-derived DCs to the NKL-code, showing that VENTX is expressed in cDCs but not in pDCs and forms part of a cDC-specific gene regulatory network operating in DC differentiation and function.

Dectin-2 Regulates the Effector Phase of House Dust Mite–Elicited Pulmonary Inflammation Independently from Its Role in Sensitization

The myeloid C-type lectin receptor Dectin-2 directs the generation of Th2 and Th17 immune responses to the house dust mite Dermatophagoides farinae through the generation of cysteinyl leukotrienes and proinflammatory cytokines, respectively, but a role for Dectin-2 in effector phase responses has not been described. In this study, we demonstrate that administration of the Dectin-2 mAb solely at the time of D. farinae challenge abrogated eosinophilic and neutrophilic inflammation in the bronchoalveolar lavage fluid and Th1, Th2, and Th17 inflammation in the lung of previously sensitized mice. Furthermore, Dectin-2 null mice (Clec4n(-/-)) sensitized with the adoptive transfer of D. farinae-pulsed wild-type (WT) bone marrow-derived dendritic cells (DCs) also had less D. farinae-elicited pulmonary inflammation, supporting an effector function for Dectin-2. The protection from pulmonary inflammation seen with the Dectin-2 mAb or in Clec4n(-/-) mice was associated with little or no reduction in lung-draining lymph node cells or their cytokine production and with no reduction in serum IgE. WT and Clec4n(-/-) mice recipients, sensitized with D. farinae-pulsed WT bone marrow-derived DCs, had comparable levels of D. farinae-elicited IL-6, IL-23, TNF-α, and cysteinyl leukotrienes in the lung. By contrast, D. farinae-elicited CCL4 and CCL8 production from pulmonary CD11c(+)CD11b(+)Ly6C(+) and CD11c(+)CD11b(+)Ly6C(-)CD64(+) monocyte-derived DCs was reduced in Clec4n(-/-) recipients. Addition of CCL8 at the time of D. farinae challenge abrogated the protection from eosinophilic, neutrophilic, and Th2 pulmonary inflammation seen in Clec4n(-/-) recipients. Taken together, these results reveal that Dectin-2 regulates monocyte-derived DC function in the pulmonary microenvironment at D. farinae challenge to promote the local inflammatory response.

Regulation on function and maturation of dendritic cells from systemic lupus erythematosus patients by triptolide

Objective To explore the mechanism of immunomodulatory activity of triptolide on systemic lupus erythematosus(SLE) patients peripheral blood mononuclear cells( PBMC)-derived dendritic cells (DCs).Methods SLE -derived DCs were sorted by flow cytometry from peripheral blood mononuclear cells,then incubated with triptolide (0,5,10,30 μg/L ).After 24 h,we collected the supernatants and then detected the concentration of IFN-α,IL-6,TNF-α using ELISA.After 5 d,the cultrural cells were collected and CD11c,CD80,CD86 expression of DCs were analyzed by flow cytometry,we observed the morphology of DC by light microscope and electron microscope scanning.Results Triptolide -treated DCs from SLE patients with active and stable disease secreted lower level of IFN-α,IL-6,TNF-α,triptolide could inhibit DCs differentiation,which displayed more immature morphology and immunophenotypes than untreatedDCs.Conclusion Triptolide could decrease the immune function of DCs from SLE,inhibit differentiation and maturation of DCs. Key words: Triptolide; Systemic lupus erythematosus; Dendritic cells; Maturation

Excretory-secretory products (ESP) from Fasciola hepatica induce tolerogenic properties in myeloid dendritic cells

Fasciola hepatica is a helminth trematode that migrates through the host tissues until reaching bile ducts where it becomes an adult. During its migration the parasite releases different excretory-secretory products (ESP), which are in contact with the immune system. In this study, we focused on the effect of ESP on the maturation and function of murine bone marrow derived-dendritic cells (DC). We found that the treatment of DC with ESP failed to induce a classical maturation of these cells, since ESP alone did not activate DC to produce any cytokines, although they impaired the ability of DC to be activated by TLR ligands and also their capacity to stimulate an allospecific response. In addition, using an in vitro ovalbumin peptide-restricted priming assay, ESP-treated DC exhibited a capacity to drive Th2 and regulatory T cell (Treg) polarization of CD4 + cells from DO11.10 transgenic mice. This was characterized by increased IL-4, IL-5, IL-10 and TGF-β production and the expansion of CD4 +CD25 +Foxp3 + cells. Our results support the hypothesis that ESP from F. hepatica modulate the maturation and function of DC as part of a generalized immunosuppressive mechanism that involves a bias towards a Th2 response and Treg development.

Niflumic acid renders dendritic cells tolerogenic and up-regulates inhibitory molecules ILT3 and ILT4

Niflumic acid is a member of non-steroidal anti-inflammatory agents, from which aspirin was recently shown to inhibit maturation of human-monocyte derived dendritic cells (DCs). DCs are crucial regulators of the immune response, capable of inducing immunity as well as tolerance. In our in vitro study we showed a tolerogenic effect of NFA on phenotype and function of LPS-matured monocyte-derived DCs. Different drug concentrations dose-dependently down-regulated the expression of co-stimulatory molecules, particularly CD80 and lowered the expression of dendritic cell marker CD1a. Opposingly, the expressions of two inhibitory surface molecules, associated with tolerogenic DCs, immunoglobulin-like transcripts (ILT)3 and ILT4 were induced in treated DCs. The levels of TNFα production by NFA-treated DCs did not change significantly compared to controls, whereas the IL-12p70 and IL-10 production was completely abrogated at higher drug concentrations. However, at lower drug concentrations, the production of IL-12p70 was increased. There were no significant differences in the uptake of FITC labeled dextran by treated DCs compared to untreated cells. In allogeneic cultures with whole CD4 + T cells, dendritic cells differentiated in the presence of NFA appeared poor stimulators of CD4 + T-cell proliferation, even compared to immature DCs (iDCs). These results indicate the immunosuppressive properties of NFA, which may be therapeutically useful in controlling chronic immune and/or inflammatory diseases, by modulating DC characteristics towards tolerogenic DCs.

2,3,7,8-Tetrachlorodibenzo- p-dioxin modulates functional differentiation of mouse bone marrow-derived dendritic cells : Downregulation of RelB by 2,3,7,8-tetrachlorodibenzo- p-dioxin

We have previously shown that benzo( a)pyrene inhibits the growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DCs) [Hwang, J.A., Lee, J.A., Cheong, S.W., Youn, H.J., Park, J.H., 2007. Benzo( a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells. Downregulation of RelB and eIF3 p170 by benzo( a)pyrene. Toxicol. Lett. 169, 82–90]. Since the toxic effects of benzo( a)pyrene are aryl hydrocarbon receptor (AhR)-dependent, we examined the effects of the very potent AhR agonist 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the growth and functional differentiation of mouse BM-derived DCs. Ten nanomolars of TCDD had significant effects on functional differentiation of mouse DCs derived from BM cultured in the presence of GM-CSF and IL-4. The yields of DCs, flow-cytometrically analyzed for co-expression of CD11c/MHCII or CD11c/CD86, were reduced for TCDD-treated cultures, but TCDD itself had no effect on the growth of BM. DCs from TCDD-treated cultures expressed higher levels of MHCII and CD86, whereas expression of CD11c was reduced, compared with vehicle-treated cultures. Production of IL-10, but not IL-12, by the DCs from TCDD-treated cultures was decreased. Allogeneic T-cell stimulating ability of TCDD-treated DCs was increased compared to control DCs. The effects of TCDD were dependent on aryl hydrocarbon receptor (AhR), because α-naphthoflavone, an AhR antagonist, suppressed the effects of TCDD on IL-10 production and T-cell stimulating ability. RT-PCR revealed the downregulation of RelB, a transcription factor necessary for DCs differentiation and function. Taken together, although benzo( a)pyrene and TCDD exert their effects via binding to AhR, their effects on the growth and functional differentiation of bone marrow-derived DCs are different.

Lead effects on development and function of bone marrow-derived dendritic cells promote Th2 immune responses

Although lead (Pb) has significant effects on the development and function of macrophages, B cells, and T cells and has been suggested to promote allergic asthma in mice and humans, Pb modulation of bone marrow (BM)-derived dendritic cells (DCs) and the resultant DC effects on Th1 and Th2 development have not been examined. Accordingly, we cultured BM cells with murine granulocyte macrophage-colony stimulating factor (mGM-CSF) ± PbCl 2. At day 10, culture supernatant (SN) and non-adherent cells were harvested for analysis. Additionally, day 10 non-adherent BM-DCs were harvested and recultured with mGM-CSF + LPS ± Pb for 2 days. The day 10 Pb exposure significantly inhibited BM-DC generation, based on CD11c expression. Although fewer DCs were generated with Pb, the existing Pb-exposed DCs had significantly greater MHC-II expression than did the non-Pb-exposed DCs. However, these differences diminished upon LPS stimulation. After LPS stimulation, CD80, CD86, CD40, CD54, and MHC-II were all up-regulated on both Pb-DCs and DCs, but Pb-DCs expressed significantly less CD80 than did DCs. The CD86:CD80 ratio suggests a Pb-DC potential for Th2 cell development. After LPS stimulation, IL-6, IL-10, IL-12 (p70), and TNF-α levels significantly increased with both Pb-DCs and DCs, but Pb-DCs produced significantly less cytokines than did DCs, except for IL-10, which further supports Pb-DC preferential skewing toward type-2 immunity. In vitro studies confirm that Pb-DCs have the ability to polarize antigen-specific T cells to Th2 cells. Pb-DCs also enhanced allogeneic and autologous T cell proliferation in vitro, and in vivo studies suggested that Pb-DCs inhibited Th1 effects on humoral and cell-mediated immunity. The Pb effect was mainly on DCs, rather than on T cells, and Pb's modification of DC function appears to be the main cause of Pb's promotion of type-2-related immunity, which may relate to Pb's enhanced activation of the Erk/MAP kinase pathway.

Benzo( a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells : Downregulation of RelB and eIF3 p170 by benzo( a)pyrene

In this study, we have investigated effects of benzo( a)pyrene (BP) on growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DC). 1 μM BP dramatically inhibited growth of BM cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Although little alterations in surface expression of CD11c, major histocompatibility complex (MHC II), and CD86 molecules characteristic of mature DC were induced by BP, production of cytokines including IL-12, IL-10, and TNF-α, and allogeneic T cell stimulating ability were severely impaired. Some of the effects of BP were dependent on arylhydrocarbon receptor (AhR), because α-naphthoflavone, an AhR antagonist, suppressed the effects of BP on IL-12 production and T cell stimulating ability, but not on DC proliferation. Expression of RelB, a transcription factor necessary for DC differentiation and function, and eIF3 p170, a subunit of eukaryotic translation initiation factor (eIF)3, was reduced upon BP treatment.

Generation and characterization of a novel hematopoietic progenitor cell line with DC differentiation potential

Studies of hematopoietic stem cell (HSC)-derived dendritic cells (DCs) are often limited by the rarity of HSC. To facilitate the study of DCs, we have generated a novel cell line (CR1) by retroviral Notch(IC) gene transfer into Sca1(+)ckit(+)lin- HSC. CR1 cells proliferated in vitro in the presence of recombinant interleukin-3. They maintained an immature progenitor cell phenotype and an intact karyotype. In the presence of granulocyte-macrophage colony-stimulating factor or Flt3L, CR1 cells differentiated into myeloid and plasmacytoid DCs, respectively. Functionally, CR1 cells were comparable to primary bone-marrow-derived DCs with respect to Toll-like-receptor-mediated maturation, cytokine release and capacity to induce effective antitumor immunity. CR1 cells thus provide an elegant new cellular tool to study DC development, function and preclinical DC-based immunotherapies.

Hsp90 Is Critical for the Regulation of Phenotype and Functionality of Human Dendritic Cells.

Hsp90, a molecular chaperone, plays a critical role in protein folding and transport, and thereby it modulates cellular activity. Pre-clinical data shows over-expression of Hsp90 in multiple myeloma (MM) and efficacy of Hsp90 inhibitor in myeloma has been determined in vitro. Based on these results, phase I/II trial evaluating clinical efficacy of the Hsp90 inhibitor is underway in MM. Although Hsp90 inhibitor shows significant effects on tumor cells, there is limited information concerning its effects on the immune system. The objective of this study was to evaluate the effects of Geldanamycin on activity of antigen-presenting cells. Immature and mature monocyte derived dendritic cells (DC) from normal human donors were used as the source of antigen-presenting cells in this study. Geldanamycin treatment of DC for 24 hours had no effect on cell viability (>90%), however, it led to a significant down-regulation of surface antigens associated with activation (CD86, CD80), maturation (CD83) and antigen presentation (HLA-ABC, HLA-DPQR). This decline was associated with changes in gene expression levels of these antigens, however the protein expression analyzed by % positive cells was not down-regulated with the treatment. Exposure to Hsp90 inhibitor was associated with significant decreases in IL-12 secretion (untrt vs. trt = 135 vs. 21 pg/ml), antigen uptake (MFI untrt 798 vs. MFI trt 449, Dextran-FITC), and antigen processing. These changes were associated with decline in DC function, which were demonstrated by significant decrease in Hsp90-treated DC compared to untreated DC in presentation of Tetanus Toxoid to autologous T lymphocytes (untrt vs. trt = 73 % vs. 47 %, CFSE proliferation), allogeneic T lymphocytes stimulation (untrt vs. trt = 232795 cpm vs. 116876 cpm, 3H-thymidine incorporation), and induction of IFN-g secretion from allogenic T lymphocytes (untrt vs. trt = 500 vs. 30 pg/ml). Taken together, these results show significant decline in DC function following Hsp90 inhibitor treatment. Further studies are underway using MM patient samples pre- and post-Hsp90 inhibitor treatment to understand in vivo effects on the immune system. Our pre-clinical data suggests the need to consider proper sequence of various therapeutic modalities, including immunotherapy, to optimize and improve clinical outcome.

Histone Deacetylase Inhibitors Induce Immuno-Dominant Suppression of Dendritic Cells.

We and others have recently demonstrated that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor with anti-neoplastic properties, reduces experimental acute graft-versus-host disease (GVHD). We have now investigated the mechanisms of action of two HDAC inhibitors, SAHA and ITF 2357, on allogeneic immune responses. Bone marrow derived dendritic cells (DCs) were preincubated with the HDAC inhibitors at nanomolar concentrations for 16–18 hours and stimulated with lipopolysaccharide (LPS). Pretreatment of DCs caused a significant reduction in the secretion of TNF-α, IL-12p70 and IL-6 compared to the untreated controls (P< 0.005). Similar effects were seen using human peripheral blood mononuclear cell derived DCs. Pre-treatment of both murine and human DCs also significantly reduced their in vitro stimulation of allogeneic T cells as measured by proliferation and IFN-γ production (P<0.01). We determined the in vivo relevance of these observations utilizing a mouse model where the responses of allogeneic donor bm12 T cells depended on the function of injected host B6 DCs would stimulate. Recipient Class-II −/− B6 (H-2b) received 11 Gy on day -1 and were injected with 4–5 x 106 wild type B6 DCs treated with SAHA or with media on days -1 and 0 and then transplanted with 2 x 106 T cells and 5 x 106 TCDBM cells from either syngeneic B6 or allogeneic bm12 donors. SAHA treatment of DCs significantly reduced expansion of allogeneic donor CD4+ T cells on day +7 after BMT compared to controls (P<0.05). SAHA treatment induced a similarly significant reduction in the expansion of CD8+ cells in Class I disparate [bm1→β2M−/−] model. In vitro, SAHA treatment significantly suppressed the expression of CD40 and CD80 but did not alter MHC class II expression. Surprisingly, when mixed with normal DCs at 1:1 ratio, SAHA treated DCs dominantly suppressed allogeneic T cell responses. The regulation of T cell proliferation was not reversible by addition of IL-12, TNF-α, IL-18, anti-IL-10 or anti-TGFβ, either alone or in combination. Suppression of allogeneic responses was contact dependent in trans-well experiments. To address whether the regulation of SAHA treated DCs required contact with T cells, we devised a three cell experiment where SAHA treated DCs lacked the capacity to present antigens to T cells. DCs from B6 MHC Class II deficient (H-2b) were treated with SAHA and co-cultured with wild type B6 (H-2b) DCs along with purified allogeneic BALB/c (H-2d) CD4+ T cells in an MLR. Allogeneic CD4+ T cells proliferated well, demonstrating the regulation to be dependent on contact between SAHA treated DCs and T cells. To address the in vivo relevance of this suppression, we utilized a well characterized [BALB/c →B6] mouse model of acute GVHD. Recipient B6 animals received 11Gy on day -1 and were injected with of 5 million host type SAHA treated or control DCs on days −1, 0, and +2. Mice were transplanted on day 0 with 2 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Injection of SAHA treated DCs resulted in significantly better survival (60% vs. 10%, P < 0.01) and significantly reduced serum levels of TNF-α, donor T cell expansion and histopathology of GVHD on day +7 after BMT compared to the controls. We conclue that HDAC inhibitors are novel immunomodulators that regulate DC function and might represent a novel strategy to prevent GVHD.

Comparative analysis of murine marrow–derived dendritic cells generated by Flt3L or GM-CSF/IL-4 and matured with immune stimulatory agents on the in vivo induction of antileukemia responses

Bone marrow (BM)-derived dendritic cells (DCs) cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) have been used to generate antitumor immune responses. The cytokine Flt3 ligand (Flt3L) also has been shown to generate BM DCs. We sought to determine if DCs generated by using Flt3L then matured with lipopolysaccharide (LPS) could lead to DCs with in vivo anti-acute myelogenous leukemia (anti-AML) activity. LPS and tumor necrosis factor alpha (TNF-alpha) are effective agents for maturing DCs; however, they have potential in vivo toxicities. Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpGs) are considered relatively nontoxic, potent activators of DC function and maturation in vitro and in vivo. We investigated whether CpGs would be comparable to TNF-alpha or LPS for the maturation of GM-CSF/IL-4-generated DCs. DCs cultured with GM-CSF/IL-4 and matured with TNF-alpha, LPS, or CpG produced a greater allogeneic T-cell response compared with Flt3L/LPS-generated DCs. All 4 distinct DC types were pulsed with AML-lysate and administered before tumor challenge produced an increase in the total number of splenic anti-AML-specific cytotoxic T-lymphocyte precursors and led to significantly (P < or =.0001) improved survival compared with nonvaccinated controls. GM-CSF/IL-4/LPS was superior to Flt3L/LPS for generating anti-AML effects in vivo. Whereas TNF-alpha was comparable to LPS in conferring on GM-CSF/IL-4 DCs anti-AML effects in vivo, CpGs were superior to LPS. These data have important clinical implications and are the first to show that Flt3L-generated DCs can provide antitumor protection and that nontoxic agents such as CpGs and Flt3L may be useful in the clinical development of DC vaccines.

Flt3 ligand therapy for recipients of allogeneic bone marrow transplants expands host CD8α+ dendritic cells and reduces experimental acute graft-versus-host disease

Recent evidence suggests that dendritic cells (DCs) can regulate and amplify immune responses. Flt3 ligand (FL)-derived DC function was tested as a stimulator of allogeneic lymphocytes in vitro and in vivo. Treatment of mice with FL dramatically expanded DC number, but DCs isolated from FL-treated mice (FL DCs) were poor stimulators of allogeneic T-cell responses in vitro. Further activation of FL DCs did not restore their stimulatory ability, and FL DCs did not suppress the stimulation of the allogeneic T cells by normal DCs. FL treatment significantly increased the CD8 alpha(+) DC subset, which appeared to be the reason for their poor stimulatory capacity. These observations were confirmed in vivo using a mouse model of acute graft-versus-host disease (GVHD) wherein host DCs play a critical role. FL treatment of recipients before allogeneic bone marrow transplantation dramatically suppressed donor T-cell responses to host antigens, thereby reducing GVHD mortality (P <.01). These data represent a novel strategy that alters host DCs and reduces acute GVHD.

A simplified method for growing dendritic cells from rat bone marrow.

Bone marrow (BM) derived dendritic cells (DC) are the most potent stimulator cells of primary mixed leukocyte reactions (MLR) and also serve as powerful antigen-presenting cells in the priming of CD4+ T cells in vivo. The investigation of mechanisms by which DC function, both in vivo and in vitro, requires optimal conditions for culture of these cells so that large numbers can be produced for analysis. With the availability of recombinant (r) GM-CSF, several reports have shown that it is possible to grow many DC from murine1 and human2 BM. Although murine rGM-CSF has been used in the rat to promote the survival of lymph-borne DC for up to 72 hours3, until this report cytokines have not been used to enhance the DC yield from fresh rat BM cultures.KeywordsBone MarrowDendntlc CellAdherent CellRepresentative DataCulture MaterialThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.