Maturation Of Murine Dendritic Cells Research Articles

Effects of experimental protein-containing pneumococcal preparations on maturation of murine dendritic cells

Dendritic cells as the most active and highly specialized antigen presenting cells, play a key role in initiating immune responses. Currently, generation of medications activating dendritic cells for development of anti-infective and anticancer vaccines is of highly relevance. Preparations of microbial origin are promising to augment activity of dendritic cells, because they carry innate immune ligands for Toll-like receptors. Such preparations include experimental protein-containing pneumococcal preparations, obtained from acetone-inactivated microbial mass of the deposited S. pneumoniae 6B vaccine strain No. 296, followed by aqueous extraction and separation of 30—100 kDa fraction. Dendritic cells were obtained from bone marrow cells of CBA mice (n = 15), and cultured in complete growth medium RPMI-1640 added with recombinant GM-CSF and IL-4 (Biosource, USA). On day 6, experimental protein-containing pneumococcal preparations (50 pl/ml) were administered to the cultured immature dendritic cells. Commercial TNFa (20 ng/ml, Biosource, USA) was used as a standard maturation inducer (positive control). Immunophenotyping of dendritic cells was conducted by using flow cytometry with FITC- and PE-labeled monoclonal antibodies against cell surface receptors: CD34, CD38, CD83, CD86, CD80, CD11c, MHC II, CD14, CD282 (TLR2), CD284 (TLR4), (eBioscience, USA). Studying an effect of preparations on maturation of dendritic cells revealed that morphological characteristics of dendritic cells generated by using experimental protein-containing preparations did not differ significantly between each other as well as those induced by TNFα. The cells were characterized by large sizes, oval or irregular shape, veiled cytoplasm, eccentrically located nucleus and numerous long thin protrusions. Experimental proteincontaining preparations induced in cultured dendritic cells decrease in count of CD34+ immature and TLR2/TLR4+ cells, increased count of cells expressing markers of adhesion (CD38+), activation (MHC II+), costimulation (CD80/ CD86+) and terminal differentiation (CD83+), which may evidence about events of differentiation upon dendritic cell maturation. The 30—100 kDa fraction increased count of cells expressing adhesion molecules to a greater extent than aqueous extract that more pronouncedly stimulated rise in count of dendritic cells bearing costimulatory molecules (p 0.05). The activity of the examined proteins regarding their effect on CD83+ cells was comparable. Experimental protein-containing antigens derived from pneumococcal vaccine strain were shown to induce maturation of dendritic cells from bone marrow precursors, induce a decrease in the count of TLR2 and TLR4-expressing cells accounting for activating effect on innate immune effectors.

Generation of αCD11b-CpG antibody conjugates for the targeted stimulation of myeloid cells

CpG oligonucleotides are short single-stranded synthetic DNA molecules. Upon binding to Toll-like receptor 9 (TLR9), CpG activates immune cells in humans and mice. This results in robust Th1 type immunity potentially resulting in clearance of pathogens, reduction of allergy and anti-tumor immunity. However, the effectiveness of CpG as an adjuvant depends on its administration route, with only strong effects seen when CpG is administered locally. As local administration is not always feasible, we generated conjugates to specifically deliver CpG to myeloid cells often abundantly present in tumors. For this we coupled CpG (3′-Thiol-modified phosphorothioate (PTO) CpG-ODN1826 type B (5′-tccatgacgttcctgacgtt-3′)) to monoclonal antibodies (mAbs) directed against the myeloid cell marker CD11b using maleimide-thiol coupling. The CD11b-CpG mAb (αCD11b-CpG) conjugates contained about four CpG molecules/conjugate and displayed binding and internalization characteristics similar to unconjugated CD11b mAbs (αCD11b). The αCD11b-CpG conjugates readily induced maturation of murine dendritic cells (DCs) in a TLR9-dependent manner in vitro. Following intravenous injection, αCD11b-CpG conjugates efficiently targeted CD11b+ immune cells in the blood, lymph nodes and spleen. Finally, injection of αCD11b-CpG conjugates, but not untargeted conjugates, induced maturation of CD11b+ cell subsets in vivo. In conclusion, conjugating CpG to αCD11b enabled specific targeting and activation of myeloid cells in vivo.

Polysaccharide from the Seeds of Plantago asiatica L. Protect Against Lipopolysaccharide-Induced Liver Injury.

Previous studies have proven that polysaccharide obtained from the seeds of Plantago asiatica L. (PLCP) could induce maturation of murine dendritic cells, promote defecation, and possess antioxidant activity in vitro. However, the effect of PLCP on lipopolysaccharide (LPS)-induced liver injury in mice has been rarely reported. In this study, we investigated the anti-inflammatory effect of PLCP on LPS-induced liver injury. Mice were pretreated orally with different dose of PLCP for 3 weeks. On day 22, they were injected intraperitoneally with LPS and sacrificed 12 h later. The results showed that PLCP inhibited the excessive production of tumor necrosis factor-α, interleukin (IL)-6, IL-10, IL-2, and IL-1β in mouse serum and liver. PLCP also improved glutathione peroxidase and total antioxidant capacity activities in mouse liver. In addition, PLCP inhibited nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 expression, and increased metallothionein production in mouse liver. Consequently, PLCP may possess protective effects on inflammatory associated liver injury.

Effects of crude extract of Capparis spinosa L. fruit alkaloids on the maturation of murine dendritic cells

Objective To investigate the effects of crude extract of Capparis spinosa L. fruit alkaloids (CSFA) on the maturation of murine bone marrow-derived dendritic cells (DCs). Methods CSFA was prepared and the contents were determined by high performance liquid chromatography. DCs were treated with different doses (1, 2, 3 mg/ml) of CSFA. The viability of DCs, the expression of surface molecules and the ability of phagocytosis were detected by flow cytometry. The secretion of cytokines was measured by ELISA. Western blot assay was performed to analyze the activation of key molecules in mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways. Results The results showed that CSFA alone had no significant influence on the expression of surface molecules and cytokines in DCs. However, it significantly decreased the expression of CD40, CD80, CD86 and MHC Ⅱ as well as the secretion of IL-12p40 and TNF-α that were induced by lipopolysaccharides (LPS), but increased IL-10 secretion and the ability of phagocytosis after treating DCs with both CSFA and LPS. Further, the phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and the nuclear translocation of NF-κBp65 induced by LPS were inhibited by CSFA. Conclusion CSFA could inhibit the maturation of DCs and the secretion of pro-inflammatory cytokines induced by LPS while increasing the secretion of the anti-inflammatory cytokine IL-10 and the ability of phagocytosis, which might involve MAPK and NF-κB signaling pathways. This study suggests that CSFA could be used as a potential immunosuppressant. Key words: Capparis spinosa L.; Alkaloid; Dendritic cell; Cytokine; Immunosuppression

TLR3-Induced Maturation of Murine Dendritic Cells Regulates CTL Responses by Modulating PD-L1 Trafficking.

Targeting TLR3 through formulations of polyI:C is widely studied as an adjuvant in cancer immunotherapy. The efficacy of such targeting has been shown to increase in combination with anti-PD-L1 treatment. Nevertheless, the mechanistic details of the effect of polyI:C on DC maturation and the impact on T-DC interactions upon PD-L1 blockade is largely unknown. Here we found that although DC treatment with polyI:C induced a potent inflammatory response including the production of type I interferon, polyI:C treatment of DCs impaired activation of peptide specific CD8+ T cells mainly due to PD-L1. Interestingly, we found that PD-L1 trafficking to the cell surface is regulated in two waves in polyI:C-treated DCs. One induced upon overnight treatment and a second more rapid one, specific to polyI:C treatment, was induced upon CD40 signaling leading to a further increase in surface PD-L1 in DCs. The polyI:C-induced cell surface PD-L1 reduced the times of contact between DCs and T cells, potentially accounting for limited T cell activation. Our results reveal a novel CD40-dependent regulation of PD-L1 trafficking induced upon TLR3 signaling that dictates its inhibitory activity. These results provide a mechanistic framework to understand the efficacy of anti-PD-L1 cancer immunotherapy combined with TLR agonists.

Activation of IL-6/STAT3-signaling cascade induces suppression of antigen presentation by human dendritic cells (TUM4P.917)

Abstract Immunosuppression in tumor microenvironments is one of the critical issues for cancer immunotherapy. We have been demonstrated that IL-6, a pleiotropic cytokine that regulate the growth, differentiation and survival of a variety of cells, significantly inhibited maturation of murine dendritic cells (DCs) via STAT3 activation both in vitro and in vivo. In this study, we focused on IL-6/STAT3-signaling cascade in human dendritic cells (DCs), and investigated the effects of IL-6 on antigen-presenting ability of DCs. Surface HLA class I, HLA class II, and costimulatory molecules of human DCs were evaluated after the stimulation with IL-6 in vitro. As a result, HLA-DR and CD86 expressions were significantly reduced by IL-6 treatment of human DCs. The reduction was remarkably blocked in the presence of STAT3 inhibitor. In addition, IFN-γ production by T cells after TCR-stimulation was significantly suppressed in the presence of IL-6-treated DCs compared with control DCs. Moreover, we found that activation of antigen-specific CD4+ T cells by DCs was remarkably reduced by the IL-6-treatment. In this study, we demonstrated that IL-6/STAT3-signaling cascade was one of the regulating factors for antigen presentation of DCs. From these findings suggest that IL-6/STAT3 signaling cascade regulates immunosuppressive function of human DCs, which would be a promising target for improving the effects of cancer immunotherapy.

Phenotypic and functional maturation of murine dendritic cells induced by 18 alpha- and beta-glycyrrhetinic acid

Various studies have described glycyrrhizin (GL), an active triterpenoic saponin extract of licorice roots, as an anti-inflammatory, antiviral, antimicrobial, anti-tumor and immunomodulating agent. The activity of GL has been mainly attributed to its metabolites, 18-alpha (GA) and 18-beta-glycyrrhetinic acid (GB), which their mechanism of action on the immune system cells is not clearly known. In this study, we have investigated the effects of GA and GB on the immune system by targeting dendritic cells and analyzing phenotypic and functional maturity of murine dendritic cells (DCs) after treatment with these components. Stimulation of DCs with GA and GB resulted in up-regulation of CD40, CD86 and MHC-II molecules indicating their effects on the maturation of DCs. These components induced the allogenic immunostimulatory capacity of DCs by stimulating the proliferation of T cells and production of the T helper (h)1-promoting cytokine, IL-12. They also increased the production of IFN-γ by T cells in mixed-lymphocyte reaction. In conclusion, these results indicate that GA and GB may insert their immunomodulatory effects by enhancing DC maturation and modulating Th1/Th2 response through an increase in Th1 responses, implying their beneficial in host defense against infectious diseases.

Lycium barbarum polysaccharides induce Toll-like receptor 2- and 4-mediated phenotypic and functional maturation of murine dendritic cells via activation of NF-κB

Dendritic cells (DCs) are potent antigen‑presenting cells that play pivotal roles in the initiation of primary immune responses. Lycium barbarum polysaccharides (LBPs) are known to have a variety of immunomodulatory functions. However, the cellular and molecular mechanisms underlying their therapeutic effects are poorly understood. In this study, we report that LBPs induce phenotypic and functional maturation of DCs. LBPs upregulated DC expression of I‑A/I‑E and CD11c, enhanced DC allostimulatory activity and induced IL‑12p40 production. Furthermore, the activity of LBPs on DCs was significantly reduced by treating the cells with anti‑TLR2 or anti‑TLR4 antibody prior to LBPs, indicating that both are possible receptors of LBPs. Maturation of DCs by LBPs was able to directly activate the nuclear transcription factor NF‑κB p65. The results revealed that LBP stimulation induces the phenotypic and functional maturation of DCs via TLR2- and/or TLR4-mediated NF‑κB signaling pathways.

The Trypanosoma brucei gambiense Secretome Impairs Lipopolysaccharide-Induced Maturation, Cytokine Production, and Allostimulatory Capacity of Dendritic Cells

Trypanosoma brucei gambiense, a parasitic protozoan belonging to kinetoplastids, is the main etiological agent of human African trypanosomiasis (HAT), or sleeping sickness. One major characteristic of this disease is the dysregulation of the host immune system. The present study demonstrates that the secretome (excreted-secreted proteins) of T. b. gambiense impairs the lipopolysaccharide (LPS)-induced maturation of murine dendritic cells (DCs). The upregulation of major histocompatibility complex class II, CD40, CD80, and CD86 molecules, as well as the secretion of cytokines such as tumor necrosis factor alpha, interleukin-10 (IL-10), and IL-6, which are normally released at high levels by LPS-stimulated DCs, is significantly reduced when these cells are cultured in the presence of the T. b. gambiense secretome. Moreover, the inhibition of DC maturation results in the loss of their allostimulatory capacity, leading to a dramatic decrease in Th1/Th2 cytokine production by cocultured lymphocytes. These results provide new insights into a novel efficient immunosuppressive mechanism directly involving the alteration of DC function which might be used by T. b. gambiense to interfere with the host immune responses in HAT and promote the infectious process.

Synergistic effect of methionine encephalin (MENK) combined with pidotimod(PTD) on the maturation of murine dendritic cells (DCs)

To gain new insight into the functional interaction between dendritic cells and methionine encephalin (MENK) combined with pidotimod (PTD), we have analyzed the effect of MENK plus PTD on the morphology, phenotype and functions of murine bone-marrow derived dendritic cells (BMDCs) in vitro. The maturation of BMDCs cultured in the presence of either MENK or PTD alone, or MENK in combination with PTD, was detected. The cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazinemethosulphate (MTS/PMS). The changes of BMDCs morphology were confirmed with light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The BMDCs treated with MENK combined with PTD displayed a higher expression of typical maturation markers of CD40, CD80, CD83, CD86 and MHC-IIidentified by fluorescence activated cell sorting (FACS), and stronger ability to drive T cells. The decrease of the endocytic ability was assayed by DAB kit, FITC-dextran and cellular immunohistochemistry. Finally upregulation of cytokines production of IL-12 and TNF-α was determined by ELISA. These data indicate that MENK combined with PTD could exert synergistic action on BMDC maturation.

A polysaccharide isolated from Pueraria lobata enhances maturation of murine dendritic cells

Maturation of dendritic cells (DCs) is a critical factor for initiating the immune response. However, DC maturation is usually attenuated in the tumor microenvironment, which is an important immunological problem in DC-based immunotherapy against cancer. Here, we report the effect of a polysaccharide (PLP) isolated from Pueraria lobata on phenotypic and functional maturation of DCs. Phenotypic maturation was demonstrated by increased expression of CD40, CD86, and major histocompatibility complex I/II. PLP induced functional maturation of DCs, as shown by increased production of interleukin (IL)-12, IL-1β, and tumor necrosis factor-α, decreased antigen capture capacity, and enhanced allogenic T cell stimulation. In addition, PLP activated DCs generated from C3H/HeN mice with normal TLR4, but not DCs from C3H/HeJ mice with mutated TLR4, suggesting that the TLR4 is a membrane receptor of PLP. We showed that PLP increased ERK, JNK, and p38 mitogen-activated protein kinase phosphorylation, and nuclear translocation of the nuclear factor-kappaB p65 subunit, which are signaling molecules downstream of TLR4. These results indicate that PLP induced DC maturation through TLR4 signaling.

Phenotypic and functional maturation of murine dendritic cells (DCs) induced by purified Glycyrrhizin (GL)

The aim of this study is to investigate phenotypic and functional modulation of murine dendritic cells (DCs) with use of purified Glycyrrhizin (GL). These impacts of GL on DCs both from bone marrow derived DCs and established DC cell 2.4 were assessed with conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that the purified GL induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD80, CD83 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs2.4 cells were down- regulated after treatment with GL (which occurs when phagocytosis of DCs2.4 cells were decreased). Finally, we proved that GL increased the production of IL-12, IL-10 and decreased the production of tumor necrosis factor alpha (TNF-α). These data indicated that GL could promote maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that GL could exert positive modulation on murine DCs.

Analysis of maturation of murine dendritic cells (DCs) induced by purified Ganoderma lucidum polysaccharides (GLPs)

To investigate and analyze induction of phenotypic and functional maturation of murine DCs by GLP. Both phenotypic and functional activities were assessed with use of conventional scanning electronic microscopy (SEM) for the morphology of the DCs, transmitted electron microscopy (TEM) for intracellular lysosomes inside the DC, cellular immunohistochemistry for phagocytosis by the DCs, flow cytometry (FCM) for the changes in key surface molecules, bio-assay for the activity of acid phosphatases (ACP), and ELISA for the production of pro-inflammatory cytokine IL-12. It was found that GLP induced phenotypic maturation, as evidenced by increased expression of CD86, CD40, and MHC II. Functional experiments showed the down-regulation of ACP inside the DCs, which occurs when phagocytosis of DCs decreased, and antigen presentation increased with maturation. Finally, GLP increased the production of IL-12.These data reveals that GLP promotes effective activation of murine DCs. This adjuvant-like activity may have therapeutic applications in clinical settings that require a boosting of the immune response. Therefore concluded that GLP can exert positive induction to murine DCs at the used concentration.

Modulation of phenotypic and functional maturation of murine dendritic cells (DCs) by purified Achyranthes bidentata polysaccharide (ABP)

There are a large number of interactions at molecular and cellular levels between the plant polysaccharides and immune system [18]. Plant polysaccharides present an interesting effects as immunomodulators, particularly in the induction of the cells both in innate and adaptive immune systems [2,5]. Activation of DCs could improve antitumoral responses usually diminished in cancer patients, and natural adjuvants provide a possibility of inducing this activation. ABP is a purified polysaccharide isolated from Achyranthes bidentata, a traditional Chinese medicine (TCM) [1]. The aim of this study is to investigate modulation of phenotypic and functional maturation of murine DCs by ABP. Both phenotypic and functional activities were assessed with use of conventional scanning electronic microscopy (SEM) for the morphology of the DC, transmitted electron microscopy (TEM) for intracellular lysosomes inside the DC, cellular immunohistochemistry for phagocytosis by the DCs, flow cytometry (FCM) for the changes in key surface molecules, bio-assay for the activity of acidic phosphatases (ACP), and ELISA for the production of pro-inflammatory cytokine IL-12. In fact, we found that purified ABP induced phenotypic maturation revealed by increased expression of CD86, CD40, and MHC II. Functional experiments showed the down-regulation of ACP inside DCs (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). Finally, ABP increased the production of IL-12. These data reveal that ABP promotes effective activation of murine DCs. This adjuvant-like activity may have therapeutic applications in clinical settings where immune responses need boosting. It is therefore concluded that ABP can exert positive modulation to murine DCs.

Immunoadjuvant effects of Hemagglutinating virus of Japan envelope (HVJ-E) on the inactivated H9 subtype avian influenza virus vaccine

Avian influenza viruses (AIV) of the H9 subtype cause serious health problems in chickens, resulting in great economic losses to the poultry industry worldwide. The killed vaccine (KV) against H9 subtype AIV has been widely used in China since 1998 but has been linked with side effects in chickens and only partial protection. A few studies have demonstrated the immunostimulatory effects of the Hemagglutinating virus of Japan envelope (HVJ-E) in cancer therapy. In this study, the adjuvant efficacy and the protective effects of HVJ-E, in combination with H9N2 AI KV against AIV were evaluated. The maturation of murine dendritic cells treated by HVJ-E was verified by FACS in the current experiment, then the antibody hemagglutination inhibition (HI) titers and cytokines and the post-challenge virological profiles (oropharyngeal and cloacal virus shedding) were investigated to define the immune responses in chickens. Our findings indicate that HVJ-E could induce dendritic cell (DC) maturation in mice. Injection of HVJ-E in chickens resulted in raised levels of IFN-β and IFN-γ being present in sera suggesting a stimulatory effect in these animals. The antibody responses to AIV of chickens inoculated with HVJ-E adjuvanted killed H9-AIV were higher than those of chickens inoculated with oil adjvanted H9-HIV. Furthermore, although inoculation of either HVJ-E or oil adjuvanted AIV reduced virus shedding following challenge, compared to controls, HVJ-E adjuvanted AIV was more effective in reducing shedding than oil adjuvant.

Midazolam Suppresses Maturation of Murine Dendritic Cells and Priming of Lipopolysaccharide-induced T Helper 1-type Immune Response

Dendritic cells (DCs), as antigen-presenting cells, play a key role in the induction and regulation of adaptive immune response. Midazolam is reported to have immunomodulatory properties that affect immune cells. However, the effect of midazolam on DCs has not been characterized. We examined the immunomodulatory properties of midazolam on DC-mediated immune response. After allowing murine bone marrow-derived DCs induced by granulocyte macrophage colony stimulating factor to mature, we analyzed their expression of costimulatory molecules (CD80 and CD86), major histocompatibility complex class II molecules, and the secretion of interleukin-12 p40. In vitro, we evaluated the effect of midazolam on maturing DCs in mixed cell cultures containing DCs and T cells. In vivo, we investigated the contact-hypersensitivity response. Midazolam suppressed the expression of CD80, CD86, and major histocompatibility complex class II molecules from murine DCs. Treated with midazolam, DCs also secreted less interleukin-12 p40. In mixed cell cultures with CD3-positive T cells, midazolam-treated DCs showed less propensity to stimulate the proliferation of CD3-positive T cells and the secretion of interferon-γ from CD4-positive T cells. Midazolam-treated DCs impaired the induction of contact-hypersensitivity response. Treatment with ligands for peripheral benzodiazepine receptor inhibited the up-regulation of CD80 during DC maturation. Midazolam inhibits the functional maturation of murine DCs and interferes with DC induction of T helper 1 immunity in the whole mouse. In addition, it appears that the immunomodulatory effect of midazolam is mediated via the action of midazolam on the peripheral benzodiazepine receptor.

Induction of indoleamine 2,3-dioxygenase expression via heme oxygenase-1-dependant pathway during murine dendritic cell maturation

Heme oxygenase (HO)-1 is expressed in a variety of conditions involved in the regulation of immune responses. In this study, we examined the role of HO-1 in dendritic cell (DC) maturation and expression of indoleamine 2,3-dioxygenase (IDO), a key enzyme that catalyzes the initial, rate-limiting step in tryptophan degradation. IDO deficiency led to diminished phenotypic and functional maturation of DCs in vitro and in vivo. IDO expression and DC maturation was abrogated by the HO inhibitor zinc protoporphrin, but increased by hemin, a potent inducer of HO-1. Moreover, LPS-induced HO-1 expression was mediated by an NF-kappaB-dependent pathway. Our findings provide additional insight into the immunological functions of IDO and HO-1, and suggest possible therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.

Nuclear Erythroid 2 p45-Related Factor 2 Inhibits the Maturation of Murine Dendritic Cells by Ragweed Extract

Oxidative stress plays an important role in immune regulation and dendritic cell (DC) maturation. Recent studies indicate that allergens, including ragweed extract (RWE), possess prooxidant activities, but how RWE interacts with DCs is not well understood. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a key transcription factor that regulates constitutive and coordinated induction of a battery of antioxidant genes. We hypothesized that RWE would activate DCs and that this response would be augmented in the absence of Nrf2. We generated bone marrow-derived DCs (BM-DCs) and isolated lung DCs from Nrf2(+/+) and Nrf2(-/-) mice and studied the effects of RWE on DCs in vitro. Under resting conditions, Nrf2(-/-) BM-DCs exhibited constitutively greater levels of inflammatory cytokines and costimulatory molecules than Nrf2(+/+) BM-DCs. Exposure to RWE impaired endocytic activity, significantly induced oxidative stress, and enhanced the expression of CD80, CD86, and MHCII in Nrf2(-/-) BM-DCs when compared with Nrf2(+/+) BM-DC, in association with reduced expression of Nrf2-regulated antioxidant genes. RWE significantly induced the secretion of inflammatory cytokines IL-6 and TNF-alpha in BM-DCs and lung DCs from Nrf2(-/-) mice than Nrf2(+/+) mice and significantly inhibited the secretion of IL-12 in Nrf2(+/+) BM-DCs and IL-18 in Nrf2(+/+) and Nrf2(-/-) BM-DCs. The stimulatory effects of RWE on DC activation were inhibited to varying degrees by the antioxidant N-acetyl cysteine. Our findings indicate that a defect in Nrf2-mediated signaling mechanisms alters the response of DCs to a common environmental allergen, which may contribute to the susceptibility to allergic diseases.

Effects of bee venom on the maturation of murine dendritic cells stimulated by LPS

Aim of study This study was performed to elicit the effectiveness of bee venom (BV), a traditional immunosuppressive Korean acupuncture agent, on the maturation of dendrtic cells (DCs). Materials and methods Immature dendritic cells (iDCs) were generated from mouse bone marrow cells with GM-CSF. After 10 days of initial differentiation, DCs were activated with lipopolysaccharides (LPS) for another 48h in the presence or absence of BV. Surface molecule analysis, intracytoplasmic staining of cytokines, FITC-conjugated antigen uptake, and transwell migration assays were conducted with iDCs and activated DCs. Results Up-regulation of costimulatory molecules, typical of mature DCs (mDCs) was inhibited by addition of BV. Pro-inflammatory cytokines were also found to be reduced with BV treatment in LPS-stimulated DC. A decrease in antigen uptake upon the maturation of DC was reversed in low dose BV treated mDC. In addition, BV treated mDC demonstrated reduced directional migration in response to CCL21, a lymphoid chemokine which directs mDC. Conclusions BV may have a therapeutic effect an on abnormally activated immune status, such as autoimmune rheumatoid arthritis, through an immune-modulatory effect on DC.

Factors involved in the maturation of murine dendritic cells transduced with adenoviral vector variants

Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35ΔRGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The α v-integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-α, and interferon-α in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of α v-integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes.