Human Peripheral Blood Dendritic Cells Research Articles

Mechanistic investigations of granzyme A adjuvant effects on γ 9δ 2T cells

Abstract γδ T cells are ideal effector cells against cancer and pathogens for numerous reasons: they are found in blood, mucosa, and lymphoid organs, develop memory responses independent of MHC restriction, present antigen, produce cytokines, and are cytolytic. An obstacle for the development of biomedical interventions targeting γδ T cells is the need for an adjuvant that promotes their expansion and is suitable for human use. The adjuvant routinely used, IL-2, has multiple drawbacks limiting its use for vaccination. Our lab has shown that memory γ 9δ 2T cells, induced by BCG vaccination, are expanded in vitro by M. tuberculosis, M.bovis(BCG), and our lab’s vaccine candidate 6-O-methylglucoselipopolysaccharide (mGLP), in the presence of IL-2. Additionally, the expanded γ 9δ 2T cells inhibit intracellular mycobacterial growth. This inhibition is dependent on granzyme A (GzmA) production by γ 9δ 2T cells, and GzmA alone can inhibit intracellular mycobacterial growth. We recently identified that GzmA can also promote expansion of γδ T cells, without exogenous IL-2, in response to mGLP and HMBPP, a phosphoantigen that stimulates all γ 9δ 2T cells. To identify γδ T cell adjuvants, in vitro expansion assays were performed using human peripheral blood mononuclear cells, dendritic cells, antigen, and adjuvant. The absolute numbers of expanded effector γδ T cells were calculated using flow cytometry after 7 days of culture. We identified that the ability of GzmA to promote γδ T cell expansion is dependent upon TLR4, CD14, and GzmA homodimerization, but independent of GzmA enzymatic activity. This new information has broad implications for vaccine design, promoting γδ T cell responses, and understanding the role of GzmA in infectious disease and cancer. Supported by grants from NIH (R01 AI048391) and Bill & Melinda Gates Foundation (OP1118659)

Polysaccharides from Astragalus membranaceus elicit T cell immunity by activation of human peripheral blood dendritic cells

Astragalus membranaceus is a widely used herbal medicine in Asia. It has been recognized as possessing various biological properties, however, studies on the activity of the A. membranaceus polysaccharide (AMP), a major component of A. membranaceus, on human peripheral blood dendritic cells (PBDCs) have not been thoroughly investigated. In this study, we found that AMP induced changes in dendritic morphology and the upregulation of activation marker expression and inflammatory cytokine production in human blood monocyte-derived dendritic cells (MDDCs). The AMP promoted the activation of both blood dendritic cell antigen 1+ (BDCA1+) and BDCA3+ PBDCs. AMP-induced secretion of cytokines in the peripheral blood mononuclear cells (PBMCs) was mainly due to PBDCs. Finally, activated BDCA1+ and BDCA3+ PBDCs by AMP elicited proliferation and activation of autologous T cells, respectively. Hence, these data demonstrated that AMPs could activate dendritic and T cells in human blood, and may provide a new direction for the application of AMPs in the regulation of human immunity.

Nutraceutical Apigenin regulates DC function in a RelB-dependent manner during neuroinflammation.

Abstract Apigenin, a natural flavonoid, found in several plants is known to have anti-oxidant and anti-inflammatory properties indicated by its use for centuries as a medicinal approach to treat inflammatory disorders. However, there are significant gaps in knowledge regarding its effect on dendritic cell (DC) function in maintaining an immune balance in immunospecialized locations like the central nervous system (CNS). In order to establish the potential utility of Apigenin as a therapeutic agent against neuroinflammatory diseases, we tested and found that Apigenin treatment ameliorated severity of disease progression and relapse after onset of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 and SJL mouse models of multiple sclerosis. An increased retention of DCs and other myeloid cells in the periphery correlated with decreased immune cell infiltration and reduced demyelination in the CNS in treated mice. Mechanistically, Apigenin treatment reduced RelB expression in presence of LPS in human peripheral blood DCs, which is central to DC maturation, its antigen presentation capabilities and DC-mediated T cell activation. IL-12A and IL-23, downstream pro-inflammatory targets of RelB were reduced upon Apigenin treatment in these cells. Further, RelB causes a metabolic switch in immune cells upon inflammation, which was seen as a decrease in glucose uptake and lactate production (glycolysis), and an increase in mitochondrial activity when LPS-induced DCs were treated with Apigenin. These results indicate a protective role of Apigenin against DC-regulated neurodegenerative effects through a probable RelB mediated pathway thus implicating a potential therapy for neuroinflammatory disease. Supported by grants from NIH: 1R01NS0971-47

Mycobacterium tuberculosis Rv3628 is an effective adjuvant via activation of dendritic cells for cancer immunotherapy

Tumor antigens (Ags) are weakly immunogenic and elicit inadequate immune responses, thus induction of antigen-specific immune activation via the maturation of dendritic cells (DCs) is a strategy used for cancer immunotherapy. In this study, we examined the effect of Rv3628 from Mycobacterium tuberculosis (Mtb) on activation of DCs and anti-tumor immunity in vivo. Intravenous injection of mice with Rv3628 promoted DC activation of spleen and lymph nodes. More importantly, Rv3628 also induced activation of DCs and enhanced Ag presentation in tumor-bearing mice. In mice bearing ovalbumin (OVA)-expressing tumors, combination treatment with Rv3628 and OVA peptide promoted OVA-specific T cell activation and accumulation of interferon (IFN)-γ and tumor necrosis factor (TNF)-α-producing OT-I and OT-II cells in tumor-draining lymph nodes. Moreover, three different tumor Ags in three different tumor models showed enhanced anti-tumor activity with Rv3628 as adjuvant, including inhibition of growth of OVA-expressing B16 melanoma, CT26 carcinoma, and B16 melanoma tumors, and a synergistic effect with anti-programmed cell death protein 1 (PD-1) antibody treatment. Additionally, potential application against human tumors was indicated by similar activation of human peripheral blood DCs by Rv3628. Taken together, these data demonstrate that Rv3628 could be an effective adjuvant in tumor immunotherapy via enhanced capacity of DC activation and Ag presentation.

Spermidine and spermine exert protective effects within the lung.

Asthma is a heterologous disease that is influenced by complex interactions between multiple environmental exposures, metabolism, and host immunoregulatory processes. Specific metabolites are increasingly recognized to influence respiratory inflammation. However, the role of protein‐derived metabolites in regulating inflammatory responses in the lung are poorly described. The aims of the present study were to quantify polyamine levels in bronchoalveolar lavages (BALs) from healthy volunteers and asthma patients, and to evaluate the impact of each polyamine on inflammatory responses using in vitro models and in a house dust mite (HDM)‐induced respiratory allergy model. Spermidine levels were decreased, while cadaverine levels were increased in BALs from asthma patients compared to healthy controls, using Ultra Performance Liquid Chromatography (UPLC). Both spermine and spermidine inhibit lipopolysaccharide (LPS)‐induced cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) in vitro. In addition, oral gavage with spermine or spermidine modulate HDM‐induced cell infiltration, cytokine secretion, and epithelial cell tight junction expression in murine models. Spermidine also reduces airway hyper‐responsiveness. These results suggest that modulation of polyamine metabolism, in particular spermidine, is associated with respiratory inflammation and these molecules and pathways should be further explored as biomarkers of disease and potential targets for novel therapies.

Comparison of human peripheral blood dendritic cell activation by four fucoidans

Brown seaweed is an important source of fucoidan, which displays immunomodulatory effects by activating various immune cells. However, these effects of fucoidans from various sources of brown seaweed have not yet been explored in human blood dendritic cells. We studied fucoidans extracted from Ecklonia cava, Macrocystis pyrifera, Undaria pinnatifida, and Fucus vesiculosus for their effects on human monocyte-derived dendritic cells (MODC) and human peripheral blood DC (PBDC) activation. Ecklonia cava fucoidan (ECF) strongly upregulated co-stimulatory molecules, major histocompatibility complex class I and II, and the production of proinflammatory cytokines in MODCs and PBDCs compared to those by the other three fucoidans. Moreover, ECF elicited the strongest effect in the induction of syngeneic T cell proliferation and IFN-γ production compared to those of other fucoidans. These results suggest that ECF could be a suitable candidate molecule for enhancing immune activation in humans compared to that with the other three fucoidans.

Polysaccharide from Astragalus membranaceus promotes the activation of human peripheral blood and mouse spleen dendritic cells.

Astragalus membranaceus (A. membranaceus) is a widely used traditional herb in China and Korea. A. membranaceus polysaccharides (AMP), which make up a major part of the root extract, have been shown to modulate immune modulations, especially activation of bone marrow-derived dendritic cells (BMDCs) and T cells. However, the immune stimulatory effect of AMP in the mouse in vivo and human peripheral blood DCs (PBDCs) has not been well investigated. In this study, we found that intravenous (i.v.) injection of AMP in C57BL/6 mice induced remarkable elevations in co-stimulatory and MHC class I and II molecule levels in the splenic DCs and its subsets. The stimulatory effect of DCs by AMP was elevated 6 h after treatment, which rapidly decreased 18 h after injection. Furthermore, AMP promoted intracellular production of pro-inflammatory cytokines in spleen DC subsets, which contributed elevation of serum cytokine levels. Finally, the AMP promoted PBDC activation. Thus, these results demonstrate that AMP can be used as an immune stimulatory molecules in human and mouse.

Human Peripheral Blood Dendritic Cell and T Cell Activation by Codium fragile Polysaccharide.

Natural polysaccharides exhibit an immunostimulatory effect with low toxicity in humans and animals. It has shown that polysaccharide extracted from Codium fragile (CFP) induces anti-cancer immunity by dendritic cell (DC) activation, while the effect of CFP has not examined in the human immune cells. In this study, we found that CFP promoted the upregulation of CD80, CD83 and CD86 and major histocompatibility complex (MHC) class I and II in human monocyte-derived dendritic cells (MDDCs). In addition, CFP induced the production of proinflammatory cytokines in MDDCs. Moreover, CFP directly induced the activation of Blood Dendritic Cell Antigen (BDCA)1+ and BDCA3+ subsets of human peripheral blood DCs (PBDCs). The CFP-stimulated BDCA1+ PBDCs further promoted activation and proliferation of syngeneic CD4 T cells. The CFP-activated BDCA3+ PBDCs activated syngeneic CD8 T cells, which produced cytotoxic mediators, namely, cytotoxic T lymphocytes. These results suggest that CFP may be a candidate molecule for enhancing immune activation in humans.

Mitomycin C Induces Immunogenic Cell Death Biomarkers In Human Bladder Tumors

IntroductionStandard‐of‐care for non‐muscle invasive bladder cancer is complete transurethral tumor resection followed by intravesical therapy with BCG, mitomycin C (MMC), gemcitabine, or other agents. Tumor recurrence is common and occurs in 40% to 80% of patients, with 10–20% progressing to muscle‐invasive disease. Earlier studies have shown selected cytotoxic agents induce immunogenic cell death (ICD) that promotes tumor response to immunotherapy and thereby offers the opportunity of developing synergistic combinations of ICD‐inducing cytotoxics with immune checkpoint blockers. The current qualifications for an ICD‐inducing cytotoxic include induction of DAMPs (danger‐associated molecular patterns) in vitro and vaccination with cytotoxic‐treated tumor cells in immunocompetent mice. Under these criteria, MMC is considered a poor ICD inducer. The present study tested the hypothesis that MMC can induce ICD in a more clinically relevant model, i.e., 3D histocultures of bladder tumors obtained from patients.MethodsWe evaluated the ability of MMC to induce two ICD biomarkers (i.e., ecto‐CRT (calreticulin) and release of HMGB1 from nucleus) and/or dendritic cell (DC) maturation in 3 bladder cancer cell lines with different differentiation status (RT4, T24, and TCCSUP), and 21 MMC‐treated human bladder tumor histocultures including 11 archived paraffin‐embedded tumors and 10 freshly obtained surgical tumor specimens. The methods included immunocytochemical staining flow/immune‐cytometry, immunoblotting, and immunohistochemical staining and quantitative imaging. Co‐cultures of human peripheral blood DC with MMC‐treated tumor cells (48 h) were used to detect DC maturation markers (CD83 and HLA‐DR).ResultsOver 85% of human bladder tumors (7/10 fresh tumors and 11/11 archived tumors) showed ecto‐CRT and/or HMGB1 release upon treatments with MMC at clinically achievable concentrations (1 and 3 μM). Induction of ICD markers by MMC was time‐dependent (peaked at 18 h) and increased with tumor grade (TCCSUP (grade 4) >T24 (grade 3)>RT4 (grade 1) (3 experiments, P<0.01, 2‐way ANOVA). HMGB1 release was increased in three cell lines with a dose‐dependent manner after 48 h MMC treatment (2 experiments, p value<0.05, 2‐way ANOVA). MMC further induced DC maturation in TCCSUP cells but not RT4 cells.ConclusionOur results indicate MMC induced ICD biomarkers and/or DC maturation in cultured human tumor cells and histocultures of human bladder tumors; the MMC effects were dependent on time, tumor grade and culture conditions.Support or Funding InformationSupported in part by Mosier Endowed Chair in Pharmaceutical Sciences at University of Oklahoma, Albert Charitable Trust, 1R01CA163015 from National Cancer Institute, and University of Kansas Cancer Center P30‐CA168524‐03.

Escherichia coli adhesion portion FimH functions as an adjuvant for cancer immunotherapy

Induction of antigen-specific immune activation by the maturation of dendritic cells (DCs) is a strategy used for cancer immunotherapy. In this study, we find that FimH, which is an Escherichia coli adhesion portion, induces toll-like receptor 4-dependent and myeloid differentiation protein 2-independent DC maturation in mice in vivo. A combined treatment regimen with FimH and antigen promotes antigen-specific immune activation, including proliferation of T cells, production of IFN-γ and TNF-α, and infiltration of effector T cells into tumors, which consequently inhibits tumor growth in mice in vivo against melanoma and carcinoma. In addition, combined therapeutic treatment of anti-PD-L1 antibodies and FimH treatment efficiently inhibits CT26 tumor growth in BALB/c mice. Finally, FimH promotes human peripheral blood DC activation and syngeneic T-cell proliferation and activation. Taken together, these findings demonstrate that FimH can be a useful adjuvant for cancer immunotherapy.

Human Miscarriage Is Associated With Dysregulations in Peripheral Blood-Derived Myeloid Dendritic Cell Subsets.

Dendritic cells (DC) are critically involved in decisions related to the acceptance or rejection of the foreign fetal antigens by the maternal immune system. However, particularly for human peripheral blood DCs (PBDC), available literature is rather inconsistent and the factors regulating these cells are ill-defined. Here, we investigated the phenotype and functionality of different human PBDC subsets during normal and pathologic pregnancies and studied an involvement of human chorionic gonadotropin (hCG) in PBDC regulation. Peripheral blood samples were obtained from normal pregnant women in all three trimesters, from first trimester miscarriage patients and from healthy non-pregnant women. Samples were analyzed for plasma hCG levels, for regulatory T (Treg) cell numbers, for frequencies of total and mature plasmacytoid (PDC) and myeloid (MDC1 and MDC2) PBDC subsets and for their cytokine secretion. In vitro assays, culturing PDC, MDC1 or MDC2 in the presence of two trophoblast cell lines, placenta explant supernatants or two hCG preparations were performed. The Treg-inducing capability of hCG- or non-hCG-treated stimulated MDC1 was assessed. Total and mature MDC1 and MDC2 frequencies increased during the first and second trimester of normal pregnancy, respectively. Miscarriage was associated with a reduced MDC1 and an increased MDC2 activation profile. PDC were not altered neither during normal pregnancy progression nor during miscarriage. In vitro, the culture of isolated PBDC subsets in the presence of placenta-derived factors impaired the maturation of MDC1 and differentially affected PDC maturation. An inhibitory effect on MDC1 and PDC maturation was also proven for the urine-derived hCG preparation. Finally, we observed a Treg cell elevation during early normal pregnancy that was not present in miscarriages. Stimulated MDC1 induced Treg cells in vitro, however, hCG was not involved in this process. Our findings suggest that during normal pregnancy PBDC subsets are differentially regulated dependent on gestational age. Miscarriage seems to be associated with dysregulations in the myeloid PBDC subsets and with disturbances in Treg cell frequencies. Moreover, our results propose an interdependency between MDC1 and Treg cells during early pregnancy. hCG, although shown to impair MDC1 maturation, does not seem to be a key regulator of PBDC alterations during pregnancy.

Study inhibition and apoptosis of peripheral blood dendritic cells hybridize tumor vaccine induced systematic immune effector cells to the BxPC-3 in vitro

Objective To observe the inhibition and apoptosis effects of systematic immune effector cells (SIECs) induced by increased expansion of normal human peripheral venous blood dendritic cells (DCs) hybridoma vaccine on orthotopic pancreatic cancer cell line (BxPC-3) in vitro. Methods The peripheral blood mononuclear cells (PBMCs) of healthy human were obtained by gradient centrifugation of lymphocyte separation fluid, and DCs and DCs derived monocytes cells (SIECs) were obtained by adherent method. DCs and SIECs were respectively cultured with granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-4, tumor necrosis factor-α (TNF-α), IL-2, BxPC-3 tumor specific antigen (pc-3tsa). After five days, DCs were collected and counted. BxPC-3TAA-sensitized DCs (referred to as sensitive DCs), and DCs were fused with BxPC-3 by PEG+ 10% dimethylsurfoxide (DMSO) with 10∶1, 5∶1, 1∶5 to obtain sensitive DCs-BxPC-3 and DCs-BxPC-3 fusion tumor vaccines. On the 7th day, SIECs were respectively mixed with sensitive DCs-BxPC-3, DCs-BxPC-3, sensitive DCs, and DCs for 1 days in a 40∶1 mixture. The mixed cultured cells were collected, and the Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay and cell counting kit-8 (CCK-8) toxicity assay were performed on BxPC-3 in the mixed target SIECs∶BxPC3=10∶1. Results The sensitives DCs, DCs and BxPC-3 were fused, and the fusion effect was the best when the ratio was 1∶1, and the fusion rate was 40.0%. The CCK-8 assay showed that the inhibition rates of BxPC-3 induced by SIECs and SIECs by sensitive DCs-BxPC-3, DCs-BxPC-3, sensitive DCs and DCs were 53.1%, 45.3% and 78.8%, respectively. The Annexin V-FITC/PI assay showed that apoptosis rates of sensitive DCs-BxPC-3, DCs-BxPC-3, sensitive DCs, DCs and SIECs were 24.17%, 11.43%, 85.87% and 29.94%. Conclusion Tumor antigen sequential sensitization DCs have a superimposed effect on antigen acquisition, which can play a better anti-tumor effect. Early DCs fusion tumor vaccine can induce SIECs to inhibit tumor cells to a certain extent. Key words: Dendritic cells; Sequential sensitization; Pancreatic cancer; Tumor vaccine; Cell inhibition; Apoptosis

Activation of Human Dendritic Cells by Ascophyllan Purified from Ascophyllum nodosum.

In our previous study, we showed that ascophyllan purified from Ascophyllum nodosum treatment promotes mouse dendritic cell (DC) activation in vivo, further induces an antigen-specific immune response and has anticancer effects in mice. However, the effect of ascophyllan has not been studied in human immune cells, specifically in terms of activation of human monocyte-derived DCs (MDDCs) and human peripheral blood DCs (PBDCs). We found that the treatment with ascophyllan induced morphological changes in MDDCs and upregulated co-stimulatory molecules and major histocompatibility complex class I (MHC I) and MHC II expression. In addition, pro-inflammatory cytokine levels in culture medium was also dramatically increased following ascophyllan treatment of MDDCs. Moreover, ascophyllan promoted phosphorylation of ERK, p38 and JNK signaling pathways, and inhibition of p38 almost completely suppressed the ascophyllan-induced activation of MDDCs. Finally, treatment with ascophyllan induced activation of BDCA1 and BDCA3 PBDCs. Thus, these data suggest that ascophyllan could be used as an immune stimulator in humans.

Effect of Endomorphin-1 on Maturation and Expression of TLR4 in Peripheral Blood Dendritic Cells Induced by High Glucose

To investigate the effects of endomorphin-1 (EM-1) on the maturation phenotype, cytokine secretion, T cell proliferation and TLR4 expression in human peripheral blood dendritic cells (PBDCs) stimulated and induced by high glucose, and to explore the regulatory mechanism of EM-1 on DC immune function. Peripheral blood mononuclear cells (PBMNCs) were induced into immature dendritic cells (imDCs). The high glucose was used as the stimulating factor, and the EM-1 was used as the interventional factor. Then, the experiments were divided into normal glucose group (NG group), high glucose group (HG group), high glucose plus EM-1 group (EM group) and high glucose plus EM-1 and naloxone group (Nal group), respectively. The PBDC's phenotype changes were detected by flow cytometry; ELISA was used to detect the changes of cytokines secreted by PBDCs co-cultured with autologous lymphocytes; CFSE was used to detect the proliferation of T lymphocytes. TLR4 expression on PBDC surface was detected by RT-PCR. Compared with HG group, the expression of PBDC surface molecules CD86, CCR7 and CD36 was up-regulated in EM group (P<0.01), while the change of CD83 expression was not statistically significant. However, IL-12 and IL-10 secreted by PBDCs and the proliferation index of T-lymphocytes stimulated by PBDCs were both decreased in EM group. Compared with EM group, the expression of CD86, CCR7 and CD36 was decreased in Nal group (P<0.01), while the expression of CD83 was almost unchanged (P>0.05). T-lymphocyte proliferation index was increased very significantly in Nal group (P<0.01). The gray ratio of TLR4 in HG group was higher than that in NG group, while the gray ratio in EM group's was very significantly lower than that in HG group's (P<0.01). These results indicate that the high glucose can promote the expression of PBDC TLR4, while the EM-1 inhibits the expression of TLR4. EM-1 up-regulates the expression of PBDC surface molecules CD86, CCR7 and CD36 stimulated and induced by high glucose, but inhibites the induction of PBDC to maturity by high glucose. And the secreted inflammatory cytokines IL-12 and IL-10 inhibites the proliferation of T lymphocytes derived from PBDCs, while naloxone inhibites the effect of EM-1. EM-1 inhibites the expression of TLR4 on PBDC surface induced by high glucose.

BLT1 in dendritic cells promotes Th1/Th17 differentiation and its deficiency ameliorates TNBS-induced colitis.

Leukotriene B4 (LTB4) synthesis is enhanced in the colonic mucosa in patients with inflammatory bowel disease (IBD). BLT1, a high-affinity receptor for LTB4, exhibits no effect on the progression of dextran sodium sulfate (DSS)-induced colitis, which mostly relies on innate immunity. Here, we reported that BLT1 regulates trinitrobenzene sulfonic acid (TNBS)-induced colitis, which reflects CD4+ T-cell-dependent adaptive immune mechanisms of IBD. We found that BLT1 signaling enhanced the progression of colitis through controlling the production of proinflammatory cytokines by dendritic cells (DCs) and modulating the differentiation of Th1 and Th17. BLT1-/- mice displayed an alleviated severity of TNBS-induced colitis with reduced body weight loss and infiltrating cells in the lamina propria. BLT1 deficiency in DCs led to reduced production of proinflammatory cytokines, including IL-6, TNF-α, and IL-12, and these results were further confirmed via treatment with a BLT1 antagonist. The impaired cytokine production by BLT1-/- DCs subsequently led to reduced Th1 and Th17 differentiation both in vitro and in vivo. We further performed a conditional DC reconstitution experiment to assess whether BLT1 in DCs plays a major role in regulating the pathogenesis of TNBS-induced colitis, and the results indicate that BLT1 deficiency in DCs also significantly reduces disease severity. The mechanistic study demonstrated that BLT1-regulated proinflammatory cytokine production through the Gαi βγ subunit-phospholipase Cβ (PLCβ)-PKC pathway. Notably, we found that treatment with the BLT1 antagonist also reduced the production of proinflammatory cytokines by human peripheral blood DCs. Our findings reveal the critical role of BLT1 in regulating adaptive immunity and TNBS-induced colitis, which further supports BLT1 as a potential drug target for adaptive immunity-mediated IBD.

Nutraceutical Apigenin regulates DC function in a RelB-dependent manner during neuroinflammation

Abstract Apigenin, a natural flavonoid, found in several plants is known to have anti-oxidant and anti-inflammatory properties indicated by its use for centuries as a medicinal approach to treat inflammatory disorders. However, there are significant gaps in knowledge regarding its effect on dendritic cell (DC) function in maintaining an immune balance in immunospecialized locations like the central nervous system (CNS). In order to establish the potential utility of Apigenin as a therapeutic agent against neuroinflammatory diseases, we tested and found that Apigenin treatment ameliorated severity of disease progression and relapse after onset of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 and SJL mouse models of multiple sclerosis. An increased retention of DCs and other myeloid cells in the periphery correlated with decreased immune cell infiltration and reduced demyelination in the CNS in treated mice. Mechanistically, Apigenin treatment reduced RelB expression in presence of LPS in human peripheral blood DCs, which is central to DC maturation, its antigen presentation capabilities and DC-mediated T cell activation. IL-12A and IL-23, downstream pro-inflammatory targets of RelB were reduced upon Apigenin treatment in these cells. Further, RelB causes a metabolic switch in immune cells upon inflammation, which was seen as a decrease in glucose uptake and lactate production (glycolysis), and an increase in mitochondrial activity when LPS-induced DCs were treated with Apigenin. These results indicate a protective role of Apigenin against DC-regulated neurodegenerative effects through a probable RelB mediated pathway thus implicating a potential therapy for neuroinflammatory disease.

Human Peripheral Blood Mononuclear Cell Function and Dendritic Cell Differentiation Are Affected by Bisphenol-A Exposure.

Environmental pollutants, including endocrine disruptor chemicals (EDCs), interfere on human health, leading to hormonal, immune and metabolic perturbations. Bisphenol-A (BPA), a main component of polycarbonate plastics, has been receiving increased attention due to its worldwide distribution with a large exposure. In humans, BPA, for its estrogenic activity, may have a role in autoimmunity, inflammatory and allergic diseases. To this aim, we assessed the effect of low BPA doses on functionality of human peripheral blood mononuclear cells (PBMCs), and on in vitro differentiation of dendritic cells from monocytes (mDCs). Fresh peripheral blood samples were obtained from 12 healthy adult volunteers. PBMCs were left unstimulated or were activated with the mitogen phytohemagglutinin (PHA) or the anti-CD3 and anti-CD28 antibodies and incubated in presence or absence of BPA at 0.1 and 1nM concentrations. The immune-modulatory effect of BPA was assessed by evaluating the cell proliferation and the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-13 (IL-13) secreted by PBMCs. mDCs were differentiated with IL-4 and GC-CSF with or without BPA and the expression of differentiation/maturation markers (CD11c, CD1a, CD86, HLA-DR) was evaluated by flow cytometry; furthermore, a panel of 27 different cytokines, growth factors and chemokines were assayed in the mDC culture supernatants. PBMCs proliferation significantly increased upon BPA exposure compared to BPA untreated cells. In addition, a significant decrease in IL-10 secretion was observed in PBMCs incubated with BPA, either in unstimulated or mitogen-stimulated cells, and at both 0.1 and 1nM BPA concentrations. Similarly, IL-13 was reduced, mainly in cells activated by antiCD3/CD28. By contrast, no significant changes in IFN-γ and IL-4 production were found in any condition assayed. Finally, BPA at 1nM increased the density of dendritic cells expressing CD1a and concomitantly decreased the expression of HLA-DR and CD86 activation markers. In conclusion, in humans the exposure to BPA causes on PBMCs a significant modulation of proliferative capacity and cytokine production, and on mDCs alteration in differentiation and phenotype. These immune cell alterations suggest that low dose chronic exposure to BPA could be involved in immune deregulation and possibly in the increased susceptibility to develop inflammatory and autoimmune diseases.

Effects of S. officinalis L. radix triterpene glycosides on innate immunity factors.

The effects of triterpene glycosides (saponins) extracted from Saponaria officinalis L. radices, on the cellular and humoral innate immunity factors were studied. Saponins stimulated the phagocytic, bactericidal, and adhesion activities of polymorphonuclear leukocytes. Optimal conditions of saponin treatment (dose and duration) were determined for mice. Saponins promoted the maturation of human peripheral blood dendritic cells, which was proven by high expression of CD83 (terminal differentiation marker) and CD86 (bone-stimulating molecule) and of HLA-DR and HLA-ABC molecules on the cell membrane. Saponins modulated the production of TNF-α, IL-1β, IL-4, IL-6, and IFN-γ in cultured peripheral blood intact cells. The results help to understand some mechanisms of the effects of saponins extracted from Saponaria officinalis L. radix on the cellular and humoral factors of innate immunity and demonstrate good prospects of their practical use.

237 : Human BDCA2+ BDCA3int dendritic cells are a novel functional subtype of classical plasmacytoid dendritic cell

Dendritic cells (DCs) are a heterogeneous cell population critical in regulating immune responses to pathogens and self-antigens. Human peripheral blood DCs have been broadly characterized as plasmacytoid DC (BDCA2 + ), myeloid DC1 (BDCA1 + ) and myeloid DC2 (BDCA3 + ) subsets. Our study aimed to understand whether BDCA3 + DCs from human peripheral blood, the putative functional equivalent to murine CD8a + DCs, constitute a homogeneous population of myeloid DC2 or if there are BDCA2 + BDCA3 + DCs that are functionally related to pDC. We constructed multi-color panels to phenotype lineage neg PBMC for expression of BDCA2 and BDCA3 by flow cytometric analysis. Three different lineage neg DC subsets from the total circulating PBMC were observed: BDCA2 + /3 neg (∼0.21%), BDCA2 + /3 int (∼0.22%), and BDCA2 neg /3 int/hi (∼0.17%). To assess the functional significance of the BDCA2 + /3 int DCs, we stimulated PBMC with TLR9 agonists CpG-A or HSV-1 and stained for intracellular IFN-a and TNF-a in cells expressing BDCA2 and/or BDCA3. At 6hrs, the stimulated BDCA2 + /3 int DC subset yielded more IFN-a + and TNF-a + cells than the other 2 subsets. By 8hrs, the % IFN-a + was similarly higher for BDCA2 + /3 − and BDCA2 + /3 int DC subsets than for BDCA2 − /3 int/hi DCs. All three DC subsets produced IFN-l1/3 upon 8hr HSV-1 stimulation, but more of the BDCA2 + /3 − and BDCA2 + /3 int DCs expressed IFN-l1/3 than BDCA2 − /3 int/hi DCs. IRF7, the master regulatory transcription factor of IFN production in pDCs, was constitutively expressed by the BDCA2 + /3 neg and BDCA2 + /3 int subsets but not by the BDCA2 neg subset. Uptake (“nibbling”) of cell-associated membrane from live virus-infected cells induces pDCs to produce IFN-a and mature. Both BDCA2 + /3 neg and BDCA2 + /3 int DCs preferentially nibbled HSV-infected Raji cells (with nibbling by BDCA2 + /3 int > BDCA2 + /3 neg DCs), while the BDCA2 neg /3 int/hi DC subset efficiently nibbled both uninfected and infected cells to a similar extent. Together, these results suggest that BDCA2 + /3 int DCs exhibit major functions associated with classical pDCs and are phenotypically and functionally distinct from BDCA2 neg /3 int/hi DCs.

Self-assembled amphiphilic phosphopyridoxyl-polyethylenimine polymers exhibit high cell viability and gene transfection efficiency in vitro and in vivo.

Branched polyethylenimine (bPEI) was conjugated with hydrophobic pyridoxal phosphate (PLP) in the side chain via reaction with primary amines to obtain amphiphilic phosphopyridoxyl-polyethylenimine (PPyP) polymers. These polymeric amphiphiles with a defined degree of hydrophobicity self-assembled into nanostructures, which were characterized by DLS and evaluated for their capability to condense nucleic acids and carry them into cells. Further condensation of pDNA compacted the size of the self-assembled nanostructures from 421-559 nm to 134-210 nm with zeta potentials from +20-32 mV to +18-28 mV. Conjugation of PLP with bPEI not only reduced the density of the primary amines (i.e. charge density) but also improved the cell viability of the modified polymers considerably and weakened the binding of pDNA with these polymers. Efficient unpackaging of the pDNA complexes inside the cells led to a several fold enhancement in the transfection efficiency with one of the formulations, PPyP-3/pDNA complex, among the series, exhibiting ∼4.9 to 8.2 folds higher gene delivery activity than pDNA complexes of bPEI and Lipofectamine™ in HeLa and MCF-7 cells. Flow cytometry analysis revealed a very high percentage of transfected cells by PPyP/pDNA complexes compared to pDNA complexes of bPEI and Lipofectamine™. Further, GFP-specific siRNA delivery using PPyP-3 as a vector resulted in ∼84% knockdown of the target gene expression (cf.∼54% by Lipofectamine™/pDNA/siRNA complex). Moreover, the PPyP-3/pDNA complex displayed ∼6.7 fold higher transfection efficiency than the bPEI/pDNA complex in human peripheral blood dendritic cells. Intravenous administration of PPyP-3/pGL3 complex showed the highest gene expression in spleen tissue, advocating the potential of these vectors in future gene delivery applications.