Murine Bone Marrow Dendritic Cells Research Articles

Role of the P2Y6 receptor of UDP in the modulation of murine dendritic cell functions and Th1 polarisation of the immune response

Numerous studies have demonstrated the role of uridine diphosphate (UDP) and its P2Y6 receptor in the inflammatory reaction and innate immunity. However, the importance of the P2Y6 receptor in the adaptive immune response remains unclear. In this study, we demonstrate that the P2Y6 receptor is functionally expressed in murine bone marrow dendritic cells (BMDC). UDP induced a Ca2+ transient in these cells that was decreased in P2Y6-deficient mice. UDP also increased the endocytosis of fluorescein isothiocyanate-dextran (FITC-dextran) and amplified the secretion of interleukin 12-p70 (IL-12p70) induced by CpG; these responses were abolished in P2Y6-deficient mice. In vivo experiments showed that the serum level of specific IgG2c after immunisation with ovalbumin was decreased in P2Y6-deficient mice, while the level of specific IgG1 was unchanged. These data suggest that the P2Y6-mediated effects of UDP on myeloid dendritic cells play a role in the in vivo Th1 skewing of the immune response.

β-Glucans from Trametes versicolor (L.) Lloyd Is Effective for Prevention of Influenza Virus Infection.

Coriolus versicolor (C. versicolor) is a higher fungi or mushroom which is now known by its accepted scientific names as Trametes versicolor (L.) Lloyd. Many studies have shown that β-glucans from C. versicolor have various physiological activities, including activating macrophages to protect against Salmonella infection. However, whether β-glucans have antiviral effects has not been reported. Hence, the objective of this study was to confirm whether β-glucans could boost the immune response to combat influenza virus in mouse and chick models. The results show that β-glucans induced the expression of Dectin-1, costimulatory molecules (CD80/86) and cytokines IL-6, IL-1β, IFN-β and IL-10 in murine bone marrow dendritic cells (BMDCs). In addition, orally administered β-glucans reduced weight loss, mortality and viral titers in the lungs of mice infected with influenza virus and attenuated pathological lung damage caused by the virus in the mice. Orally administered β-glucans improved survival and reduced lung viral titers in chickens infected with H9N2 avian influenza virus. These results suggest that β-glucans have a significant antiviral effect. Therefore, β-glucans could become a potential immunomodulator against influenza virus.

Expansion of T regulatory lymphocytes by murine bone marrow dendritic cells previously stimulated with Anisakis simplex larval antigens.

BACKGROUND Anisakis simplex antigens present immunomodulatory properties by the induction of tolerogenic dendritic cells (DCs) in mice.OBJECTIVES To study the capacity of DCs stimulated with A. simplex excretory-secretory (ES) or crude extract (CE) to generate Tregs. To investigate in vitro effects of antigens on the metabolic activity of splenocytes induced by LPS or CpG.METHODS Phenotypic and functional characterization of T cells co-cultured with A. simplex-pulsed DCs was performed by flow cytometry. Lymphocyte mitochondrial respiratory activity was estimated by the Alamar Blue® Assay.FINDINGS In C57BL/6J, CD4+CD25-Foxp3+ and CD8+CD25-Foxp3+ populations increased by CE-stimulated-DCs. In BALB/c, CE-stimulated-DCs caused the expansion of CD4+CD25+Foxp3+IL-10+ and CD8+CD25+Foxp3+IL-10+. IFN-γ expression raised in BALB/c CD4+CD25+ and CD4+CD25- for CE and ES, respectively. ES-stimulated-DCs increased CD4+CD25+ Foxp3+ and CD8+CD25- Foxp3+ expression in T cells. The association of ES or CE with LPS produced the increase in splenocyte activity in C57BL/6J. The association of CE with CpG decreased the proliferation caused by CpG in C57BL/6J.MAIN CONCLUSIONS A. simplex increase the frequency of Tregs, which in turn produce IL-10 and IFN-γ. The host genetic base is essential in the development of anti-Anisakis immune responses (Th2, Th1, Treg).

Immunomodulatory Protective Effects of Rb9 Cyclic-Peptide in a Metastatic Melanoma Setting and the Involvement of Dendritic Cells.

The cyclic VHCDR3-derived peptide (Rb9) from RebMab200 antibody, directed to a NaPi2B phosphate-transport protein, displayed anti-metastatic melanoma activity at 50–300 μg intraperitoneally injected in syngeneic mice. Immune deficient mice failed to respond to the peptide protective effect. Rb9 induced increased CD8+ T and low Foxp3+ T cell infiltration in lung metastases and high IFN-γ and low TGF-β in lymphoid organs. The peptide co-localized with F-actin and a nuclear site in dendritic cells and specifically bound to MIF and CD74 in a dot-blot setting. Murine bone-marrow dendritic cells preincubated with Rb9 for 6 h were treated with MIF for short time periods. The modulated responses showed stimulation of CD74 and inhibition of pPI3K, pERK, and pNF-κB as compared to MIF alone. Rb9 in a melanoma-conditioned medium, stimulated the M1 type conversion in bone marrow-macrophages. Functional aspects of Rb9 in vivo were studied in therapeutic and prophylactic protocols using a melanoma metastatic model. In both protocols Rb9 exhibited a marked anti-melanoma protection. Human dendritic cells were also investigated showing increased expression of surface markers in response to Rb9 incubation. Rb9 either stimulated or slightly inhibited moDCs submitted to inhibitory (TGF-β and IL-10) or activating (LPS) conditions, respectively. Lymphocyte proliferation was obtained with moDCs stimulated by Rb9 and tumor cell lysate. In moDCs from cancer patients Rb9 exerted immunomodulatory activities depending on their functional status. The peptide may inhibit over-stimulated cells, stimulate poorly activated and suppressed cells, or cause instead, little phenotypic and functional alterations. Recently, the interaction MIF-CD74 has been associated to PD-L1 expression and IFN-γ, suggesting a target for melanoma treatment. The effects described for Rb9 and the protection against metastatic melanoma may suggest the possibility of a peptide reagent that could be relevant when associated to modern immunotherapeutic procedures.

Bone marrow dendritic cells regulate hematopoietic stem/progenitor cell trafficking.

A resident population of dendritic cells (DCs) has been identified in murine bone marrow, but its contribution to the regulation of hematopoiesis and establishment of the stem cell niche is largely unknown. Here, we show that murine bone marrow DCs are perivascular and have a type 2 conventional DC (cDC2) immunophenotype. RNA expression analysis of sorted bone marrow DCs shows that expression of many chemokines and chemokine receptors is distinct from that observed in splenic cDC2s, suggesting that bone marrow DCs may represent a unique DC population. A similar population of DCs is present in human bone marrow. Ablation of conventional DCs (cDCs) results in hematopoietic stem/progenitor cell (HSPC) mobilization that is greater than that seen with ablation of bone marrow macrophages, and cDC ablation also synergizes with G-CSF to mobilize HSPCs. Ablation of cDCs is associated with an expansion of bone marrow endothelial cells and increased vascular permeability. CXCR2 expression in sinusoidal endothelial cells and the expression of two CXCR2 ligands, CXCL1 and CXCL2, in the bone marrow are markedly increased following cDC ablation. Treatment of endothelial cells in vitro with CXCL1 induces increased vascular permeability and HSPC transmigration. Finally, we show that HSPC mobilization after cDC ablation is attenuated in mice lacking CXCR2 expression. Collectively, these data suggest that bone marrow DCs play an important role in regulating HSPC trafficking, in part, through regulation of sinusoidal CXCR2 signaling and vascular permeability.

Evaluation of the toxicity of iron-ion irradiation in murine bone marrow dendritic cells via increasing the expression of indoleamine 2,3-dioxygenase 1.

High linear energy transfer radiation is known to deposit higher energy in tissues and cause greater toxicity compared to low-LET irradiation. Local immunosuppression is frequently observed after irradiation (IR). Dendritic cells (DCs) play important roles in the initiation and maintenance of the immune response. The dysfunction of DCs contributes to tumor evasion and growth. However, molecular mechanisms underlying the establishment of immune tolerance induced by heavy ion IR through this DC population are poorly understood. Therefore, here we report our findings on the dysfunction of bone marrow-derived dendritic cells (BMDCs) induced by 1 Gy iron ion radiation and promotions of expressions of JNK1/2/3, indoleamine 2,3-dioxygenase 1 (IDO1), p-ERK1/2 and p38/MAPK; and decrease of IDO2, MHC class II, CD40, CD80 expressions and IFN-γ and TNF-α secretion after total-body IR in mice. JNK+IDO1+ BMDCs showed up-expression of p-ERK1/2 and p-p38/MAPK, reduced expression of MHC class II and CD80, and were not able to effectively stimulate allogeneic spleen T cells. The inhibition of IDO1 expressions could partly restore the function of BMDCs. In all, our study shows that elevated JNK and IDO1 expression induced by Fe ion IR could result in dysfunction of BMDCs via p-p38/MAPK and p-ERK1/2 signal pathway, and it may represent a new mechanism in radiation-induced immune tolerance.

Role of the P2Y6 receptor of UDP in the modulation of murine dendritic cell functions and Th1 polarisation of the immune response

Numerous studies have demonstrated the role of uridine diphosphate (UDP) and its P2Y6 receptor in the inflammatory reaction and innate immunity. However, the importance of the P2Y6 receptor in the adaptive immune response remains unclear. In this study, we demonstrate that the P2Y6 receptor is functionally expressed in murine bone marrow dendritic cells (BMDC). UDP induced a Ca2+ transient in these cells that was decreased in P2Y6-deficient mice. UDP also increased the endocytosis of fluorescein isothiocyanate-dextran (FITC-dextran) and amplified the secretion of interleukin 12-p70 (IL-12p70) induced by CpG; these responses were abolished in P2Y6-deficient mice. In vivo experiments showed that the serum level of specific IgG2c after immunisation with ovalbumin was decreased in P2Y6-deficient mice, while the level of specific IgG1 was unchanged. These data suggest that the P2Y6-mediated effects of UDP on myeloid dendritic cells play a role in the in vivo Th1 skewing of the immune response.

Synthesis, Formulation, and Adjuvanticity of Monodesmosidic Saponins with Olenanolic Acid, Hederagenin and Gypsogenin Aglycones, and some C-28 Ester Derivatives.

In an attempt to discover a new synthetic vaccine adjuvant, the glycosylation of hederagenin, gypsogenin, and oleanolic acid acceptors with di‐ and trisaccharide donors to generate a range of mimics of natural product QS‐21 was carried out. The saponins were formulated with phosphatidylcholine and cholesterol, and the structures analyzed by transmission electron microscopy. 3‐O‐(Manp(1→3)Glcp)hederagenin was found to produce numerous ring‐like micelles when formulated, while C‐28 choline ester derivatives preferred self‐assembly and did not interact with the liposomes. When alone and in the presence of cholesterol and phospholipid, the choline ester derivatives produced nanocrystalline rods or helical micelles. The effects of modifying sugar stereochemistry and the aglycone on the immunostimulatory effects of the saponins was then evaluated using the activation markers MHC class II and CD86 in murine bone marrow dendritic cells. The most active saponin, 3‐O‐(Manp(1→3)Glcp)hederagenin, was stimulatory at high concentrations in cell culture, but this did not translate to strong responses in vivo.

Silenced suppressor of cytokine signaling 1 (SOCS1) enhances the maturation and antifungal immunity of dendritic cells in response to Candida albicans in vitro.

Dendritic cells (DCs) are known to play an important role in initiating and orchestrating antimicrobial immunity. Given the fact that candidiasis appears often in immunocompromised patients, it seems plausible that DCs hold the key to new antifungal strategies. One possibility to enhance the potency of DC-based immunotherapy is to silence the negative immunoregulatory pathways through the ablation suppressor of cytokine signaling suppressor 1 (SOCS1). Here, we deliver small interfering RNA (siRNA) against SOCS1 into murine bone marrow DCs, and as a consequence, we investigate the maturation/action of DCs and the subsequent T cell response after exposure to C. albicans. Our results show that the maturation of DCs (i.e., expressions of CD80, CD40, CD86, and MHC II) are significantly increased in the silenced SOCS1 treatment group after exposure to C. albicans. As a result, suppression of the SOCS1 promotes the greatest expression of IFN-γ and IL-12, and reduces IL-4 secretions, which induce CD4+ cell Th1 differentiation but inactivate Th2 cell development. The responses of IL-6 and TNF-β consist of up-regulation in the presence of C. albicans, but this is not specific to SOCS1 silencing, suggesting that these cytokines are not regulated by the SOCS1 gene in fungal infections. We find Th17 differentiation is unchanged regardless of SOCS1 inhibition. The increase in phagocytosis and killing of C. albicans in SOCS1 gene-treated DCs indicate a role for this cytokine suppressor in innate immunity as well. In conclusion, our findings support the view that SOCS1 protein is a critical inhibitory molecule for controlling cytokine response and antigen presentation by DCs, thereby regulating the magnitude of innate and adaptive immunities by generating IFN-γ-production T cells (Th1)—but not Th17—from naïve CD4+ T cells. Our study demonstrates that SOCS1 siRNA can serve as a useful vehicle to modulate the function of DCs against C. albicans infection. Electronic supplementary materialThe online version of this article (doi:10.1007/s12026-014-8562-8) contains supplementary material, which is available to authorized users.

Specific activation of dendritic cells enhances clearance of Bacillus anthracis following infection.

Dendritic cells are potent activators of the immune system and have a key role in linking innate and adaptive immune responses. In the current study we have used ex vivo pulsed bone marrow dendritic cells (BMDC) in a novel adoptive transfer strategy to protect against challenge with Bacillus anthracis, in a murine model. Pre-pulsing murine BMDC with either recombinant Protective Antigen (PA) or CpG significantly upregulated expression of the activation markers CD40, CD80, CD86 and MHC-II. Passive transfusion of mice with pulsed BMDC, concurrently with active immunisation with rPA in alum, significantly enhanced (p<0.001) PA-specific splenocyte responses seven days post-immunisation. Parallel studies using ex vivo DCs expanded from human peripheral blood and activated under the same conditions as the murine DC, demonstrated that human DCs had a PA dose-related significant increase in the markers CD40, CD80 and CCR7 and that the increases in CD40 and CD80 were maintained when the other activating components, CpG and HK B. anthracis were added to the rPA in culture. Mice vaccinated on a single occasion intra-muscularly with rPA and alum and concurrently transfused intra-dermally with pulsed BMDC, demonstrated 100% survival following lethal B. anthracis challenge and had significantly enhanced (p<0.05) bacterial clearance within 2 days, compared with mice vaccinated with rPA and alum alone.

Maturation of mouse bone marrow dendritic cells (BMDCs) induced by Laminaria japonica polysaccharides (LJP)

The seaweed Laminaria japonica has been investigated in a laboratory research for its medical significance and LJP has been purified now. The objective of present study was to look at effect of LJP on structural, phenotypic and functional maturation of murine BMDCs. The structural maturation of BMDCs induced by LJP was evaluated by transmission electron microscopy (TEM); The phenotypic maturation of BMDCs was studied by flow cytometry(FCM) and functional maturation of BMDCs was analyzed by FITC-dextran, acid phosphatase (ACP) activity and enzyme linked immunosorbent assay (ELISA). We hereby proved that LJP markedly induced maturation of BMDCs with the data of decreased the number of lysosomes, upregulated expression of CD80, CD83, CD86, CD40 and MHC II key membrane molecules on BMDCs, downregulated phagocytosis, enriched production of IL-12 and TNF-α secreted by BMDCs. Therefore it should be concluded that LJP was with strong ability to induce maturation of BMDCs. Our data provided direct evidence to suggest that LJP could be considered as an immune stimulant in improving immune handicapped situation and as a useful adjuvant in vaccine designing.

Preservation of Dendritic Cell Function during Vesicular Stomatitis Virus Infection Reflects both Intrinsic and Acquired Mechanisms of Resistance to Suppression of Host Gene Expression by Viral M Protein

Inhibition of host-directed gene expression by the matrix (M) protein of vesicular stomatitis virus (VSV) effectively blocks host antiviral responses, promotes virus replication, and disables the host cell. However, dendritic cells (DC) have the capacity to resist these effects and remain functional during VSV infection. Here, the mechanisms of DC resistance to M protein and their subsequent maturation were addressed. Flt3L-derived murine bone marrow dendritic cells (FDC), which phenotypically resemble resident splenic DC, continued to synthesize cellular proteins and matured during single-cycle (high-multiplicity) and multicycle (low-multiplicity) infection with VSV. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived myeloid DC (GDC), which are susceptible to M protein effects, were nevertheless capable of maturing, but the response was delayed and occurred only during multicycle infection. FDC resistance was manifested early and was type I interferon (IFN) receptor (IFNAR) and MyD88 independent, but sustained resistance required IFNAR. MyD88-dependent signaling contributed to FDC maturation during single-cycle infection but was dispensable during multicycle infection. Similar to FDC, splenic DC were capable of maturing in vivo during the first 24 h of infection with VSV, and neither Toll-like receptor 7 (TLR7) nor MyD88 was required. We conclude that FDC resistance to M protein is controlled by an intrinsic, MyD88-independent mechanism that operates early in infection and is augmented later in infection by type I IFN. In contrast, while GDC are not intrinsically resistant, they can acquire resistance during multicycle infection. In vivo, splenic DC resist the inhibitory effects of VSV, and as in multicycle FDC infection, MyD88-independent signaling events control their maturation.

Phenotypic and functional analysis of the modification of murine bone marrow dendritic cells (BMDCs) induced by neutral Ginseng polysaccharides (NGP)

In this study, we reveal that a neutral polysaccharide isolated from a Chinese medicinal herb, named Ginseng (Panaxgiseng C.A. Meyer), promotes maturation of BMDCs via inducing changes both inside and outside BMDCs, as well as changes of functions. These affects of NGP on BMDCs were evaluated with use of conventional scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) for morphology of BMDCs, flow cytometry (FCM) for key surface markers of BMDCs, cytochemistry assay, FITC-dextran, bio-assay for their phagocytosis and enzyme linked immunosorbent assay (ELISA) for cytokine production by BMDCs. Our results proved that NGP induced maturation of BMDCs as reflected by the downregulation of acid phosphatase (ACP) activity inside the BMDCs, which occurs when phagocytosis of BMDCs decreased, while antigen presentation increased upon maturation. These data also revealed higher expression of MHC II, CD80, CD86, CD83, CD40 and secretion of higher level of IL-12 and low level of TNF-α. Our approach suggests that NGP could therefore stimulate the maturation of murine BMDCs through a series of regulation to the BMDCs.

Maturation of murine bone marrow dendritic cells induced by acidic Ginseng polysaccharides

In this study, we report that a acidic polysaccharide (AGP) isolated from a Chinese medicinal herb, named Ginseng (Panax giseng C.A. Meyer), induces maturation of bone marrow dendritic cells (BMDCs) via concrete changes both inside and outside BMDCs. The impacts of AGP on BMDCs were assessed with use of conventional scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) for morphology, flow cytometry (FCM) for key surface molecules, cytochemistry assay, FITC-dextran, bio-assay for phagocytosis and enzyme linked immunosorbent assay (ELISA) for production of cytokines. Our results elucidated that PPS promoted maturation of BMDCs via changes as reflected by the down-regulation of acid phosphatase (ACP) activity inside the BMDCs, which occurs when phagocytosis of BMDCs to antigen decreased, while antigen presentation increased upon maturation, higher expression of key surface molecules of MHC II, CD80, CD86, CD83, and CD40, and releasing higher level of cytokines IL-12 and low level of TNF-α. Our study suggest that AGP play marked immunostimulating role on the maturation of murine BMDCs through precise regulation of phagocytosis and enzyme activities inside the BMDCs.

Lactococcus lactis subsp. cremoris FC triggers IFN-γ production from NK and T cells via IL-12 and IL-18

Lactic acid bacteria (LAB) benefit health as probiotics in a strain-dependent way. In this study, we investigated the immunomodulatory effects of Lactococcus lactis subsp. cremoris FC (LcFC) on dendritic cells (DCs), natural killer (NK) cells and T cells. LcFC induced the production of cytokines such as IL-10, IL-12, IL-6 and TNF-α from murine bone marrow DCs (BMDCs) via MyD88-dependent pathway. In comparison with the type strain L. lactis subsp. cremoris ATCC 19257, LcFC induced particularly high production of IL-12 while induction of IL-6 was moderate. Consequently, LcFC triggered IFN-γ production in splenic NK, CD8+, and CD4+ cells. Most prominent effect of LcFC on IFN-γ production was observed in NK cells, followed by CD8+ cells, which was completely inhibited by combination of neutralizing anti-IL-12 and anti-IL-18 mAbs. Moreover, oral administration of LcFC enhanced the production of IFN-γ and IL-10 from splenocytes of treated mice. These findings suggest that this LAB strain is an efficient activator of protective cellular immunity via stimulation of myeloid cells including DCs.

Cytohesin-1 controls the activation of RhoA and modulates integrin-dependent adhesion and migration of dendritic cells

AbstractAdhesion and motility of mammalian leukocytes are essential requirements for innate and adaptive immune defense mechanisms. We show here that the guanine nucleotide exchange factor cytohesin-1, which had previously been demonstrated to be an important component of beta-2 integrin activation in lymphocytes, regulates the activation of the small GTPase RhoA in primary dendritic cells (DCs). Cytohesin-1 and RhoA are both required for the induction of chemokine-dependent conformational changes of the integrin beta-2 subunit of DCs during adhesion under physiological flow conditions. Furthermore, use of RNAi in murine bone marrow DCs (BM-DCs) revealed that interference with cytohesin-1 signaling impairs migration of wild-type dendritic cells in complex 3D environments and in vivo. This phenotype was not observed in the complete absence of integrins. We thus demonstrate an essential role of cytohesin-1/RhoA during ameboid migration in the presence of integrins and further suggest that DCs without integrins switch to a different migration mode.

Local production and activation of complement up-regulates the allostimulatory function of dendritic cells through C3a–C3aR interaction

Donor cell expression of C3 enhances the alloimmune response and is associated with the fate of transplantation. To clarify the mechanism for enhancement of the immune response, we have explored the role of C3a receptor (C3aR)-ligand interaction on murine bone marrow dendritic cells (DCs). We show that DCs either lacked receptor for C3a (a C3 cleavage product) or were treated with C3aR antagonist, elicited defective T-cell priming against alloantigen expressed on the DCs. This was associated with reduced surface expression of major histocompatibility complex (MHC) and costimulatory molecules on the DCs, and with defective priming in skin allograft rejection. In addition, DCs lacking factor B were unable to generate potent T-cell responses against donor antigen, whereas lack of C4 had no detectable effect, suggesting a role for the alternative pathway contributing to allostimulation. Furthermore, therapeutic complement regulator can down-regulate DC allostimulatory function. These findings suggest that the capacity of DCs for allostimulation depends on their ability to express, activate, and detect relevant complement components leading to C3aR signaling. This mechanism, in addition to underpinning the cell-autonomous action of donor C3 on allostimulation, has implications for a wider range of immune responses in self-restricted T-cell priming.

Immunoadjuvant effects of polyadenylic:polyuridylic acids through TLR3 and TLR7

Double-stranded RNA (dsRNA) is produced upon viral infection and can activate innate immunity. Polyinosinic:polycytidylic acids [poly(I:C)] is a synthetic mimetic of dsRNA and functions through an endosomal receptor, Toll-like receptor (TLR) 3 or cytosolic receptors. Another type of dsRNA, polyadenylic:polyuridylic acids [poly(A:U)], can also act as an immune adjuvant, but it remains unclear how it exhibits its adjuvant effects. Here, we have characterized the adjuvant effects of poly(A:U). Poly(A:U) could induce both IFN-alpha and IL-12p40 from murine bone marrow dendritic cells (DCs). Poly(A:U)-induced IFN-alpha production depended on a DC subset, plasmacytoid dendritic cell (pDC), and required TLR7. IL-12p40 was also produced by poly(A:U)-stimulated pDC in a TLR7-dependent manner. In addition to pDC, conventional dendritic cell (cDC) also produced IL-12p40 in response to poly(A:U). This IL-12p40 induction resulted from two cDC subsets, CD24(high) cDC and CD11b(high) cDC in a TLR3- and TLR7-dependent manner, respectively. In vivo injection of poly(A:U) with antigen led to clonal expansion of and IFN-gamma production from antigen-specific CD8(+) T cells. Consistent with the in vitro findings, TLR3 and TLR7 were required for the clonal T-cell expansion. Notably, TLR3, rather than TLR7, was critical for generating IFN-gamma-producing CD8(+) T cells. CD8(+) T-cell responses induced by poly(A:U) were independent of type I IFN signaling. Our results demonstrate that poly(A:U) functions as an in vivo immunoadjuvant mainly through TLR3 and TLR7.

Disparity in mycobacterium tuberculosis-induced IL-12 release in dendritic cells and macrophages is due to utilization of distinct Toll-like receptors (89.35)

Abstract In the pursuit of understanding the molecular basis for a differential release of interleukin (IL)-12 between dendritic cells (DCs) and macrophages in response to Mycobacterium tuberculosis (Mtb), we first evaluated the expression of IL-12p35 and IL-12p40. The two cell types showed similar transcription levels of IL-12p35, while IL-12p40 mRNA was expressed in a rapid and enhanced manner only in murine bone-marrow DCs. An analysis of the remodeling at the nucleosome 1 of the IL-12p40 promoter also provided evidence of a rapid and extensive accessibility to the transcription factor NF-kB in DCs. Despite restoring the impaired Mtb-dependent release of IL-12, addition of interferon (IFN) 2 □ or removal of IL-10 did not affect the extent of remodeling in the IL-12 promoter of macrophages. By evaluating the IL-12p40 remodeling and release in knock-out mice models we demonstrate that DCs mainly rely on signaling from toll-like receptor (TLR)9 for their extensive Mtb induced IL-12 expression. Moreover the TLR9 ligand CpG induced extensive remodeling and release of IL-12p40 in comparison to a TLR2 agonist. Our work suggests a different handling of Mtb from the two cell types where TLR9 engagement occurs prevalently in DCs and leads to high expression of IL-12 and supports the rising opinion, which favors a role for TLR2 in Th2 type responses.

Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone marrow dendritic cells by modulating class II MHC expression

Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O 2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O 2 atmospheres and omitting 2-Me from the medium were tested upon the generation of GM-CSF expanded BMDCs. DC yield, phenotype and function were compared to BMDCs grown using conventional conditions. All cultures yielded DC subsets with CD11c + MHC II NEG, CD11c + MHC II INT, CD11c + MHC II HI expression phenotypes, classed as precursor, immature, and mature DCs (IDC, MDC). Low O 2 tensions generated significantly fewer precursor DCs, and more IDCs and MDCs. Cytometer sorted precursor DCs expressed surface class II MHC after transfer to low, but not high O 2 atmospheres. Expression of myeloid markers was similar between BMDC cultures generated in 5% O 2 or conventional conditions, and MDCs from low O 2 cultures had the morphology typical of mature myeloid DCs. IDCs and MDCs from low O 2 and conventional culture conditions were similarly potent allostimulatory APCs. The O 2 tension (but not 2-Me addition) in vitro significantly influences overall DC subset frequencies and yield, and governs DC maturation by regulating the surface class II MHC expression of GM-CSF expanded BMDC cultures.