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Background & Objectives: Scrub typhus is a chigger-borne disease caused by Orientia tsutsugamushi. The immunological reactions to O. tsutsugamushi infection are not completely understood. In this study, we investigated the response of dendritic cells (DCs) to a major 56-kDa scrub typhus antigen Sta56., Methods: Monocyte-derived human DCs were incubated with different concentrations of recombinant Sta56 and analyzed for maturation based on phagocytic capacity, the ability to induce T-cell proliferation, expression of surface markers, cytokine secretion and activation of toll-like receptor (TLR)-dependent signalling pathways., Results: Treatment of DCs with Sta56 induced cell surface expression of CD80, CD83, CD86 and MHC Class II increased the production of interleukin-12 (IL-12) p40, IL-12 p70 and IL-10 and decreased DC phagocytic capacity. Furthermore, Sta56 increased the ability of DCs to activate T-cell proliferation and interferon-γ secretion. TLR4-specific antibodies neutralized Sta56-elicited effects on DC maturation, suggesting direct interaction between Sta56 and TLR4. Moreover, Sta56 activated nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK) signalling as evidenced by decrease in Sta56-induced cytokine production and surface marker expression by specific inhibitors helenalin and SB203580, respectively, and increase in IκBα and p38 phosphorylation and NF-κB-DNA binding., Interpretation & Conclusions: Our results showed that the surface antigen of O. tsutsugamushi activated DCs through interaction with TLR4 and activation of MAPK and NF-κB signalling, suggesting Sta56 as a potential candidate molecule for the development of vaccine against scrub typhus., Competing Interests: None

Unlabelled: Influenza virus hemagglutinin (HA) N-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response. Considered essential for controlling innate and adaptive immune responses against influenza virus infections, dendritic cells (DCs) trigger proinflammatory and adaptive immune responses in hosts. In this study, we engineered Chinese hamster ovary (CHO) cell lines expressing recombinant HA from pandemic H1, H5, and H7 influenza viruses. rH1HA, rH5HA, and rH7HA were obtained as wild-type proteins or in the presence of kifunensine (KIF) or further with endo-β-N-acetylglucosaminidase-treated KIF (KIF+E) to generate single-N-acetylglucosamine (GlcNAc) N-glycans consisting of (i) terminally sialylated complex-type N-glycans, (ii) high-mannose-type N-glycans, and (iii) single-GlcNAc-type N-glycans. Our results show that high-mannose-type and single-GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70, and hIL-10 production in human DCs. Significantly higher HLA-DR, CD40, CD83, and CD86 expression levels, as well reduced endocytotic capacity in human DCs, were noted in the high-mannose-type rH1HA and single-GlcNAc-type rH1HA groups than in the complex-type N-glycan rH1HA group. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). The high-mannose-type or single-GlcNAc-type N-glycans of both avian and human HA types are more stimulatory than the complex-type N-glycans. HA-stimulated DC activation was accomplished partially through a mannose receptor(s). These results provide more understanding of the contribution of glycosylation of viral proteins to the immune responses and may have implications for vaccine development., Importance: Influenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role in the link between innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5HA, and rH7HA proteins containing distinct N-glycan patterns. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human DCs. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). High-mannose-type and single-GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering mouse and human DC activation and maturation. We believe these results provide some useful information for influenza vaccine development regarding how influenza virus HA proteins with different types of N-glycans activate DCs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Enterovirus 71 (EV71) causes seasonal epidemics of hand-foot-and-mouth disease and has a high mortality rate among young children. We recently demonstrated potent induction of the humoral and cell-mediated immune response in monkeys immunized with EV71 virus-like particles (VLPs), with a morphology resembling that of infectious EV71 virions but not containing a viral genome, which could potentially be safe as a vaccine for EV71. To elucidate the mechanisms through which EV71 VLPs induce cell-mediated immunity, we studied the immunomodulatory effects of EV71 VLPs on human monocyte-derived dendritic cells (DCs), which bind to and incorporate EV71 VLPs. DC treatment with EV71 VLPs enhanced the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR on the cell surface; increased the production of interleukin (IL)-12 p40, IL-12 p70, and IL-10 by DCs; and suppressed the capacity of DCs for endocytosis. Treatment with EV71 VLPs also enhanced the ability of DCs to stimulate naïve T cells and induced secretion of interferon (IFN)-γ by T cells and Th1 cell responses. Neutralization with antibodies against Toll-like receptor (TLR) 4 suppressed the capacity of EV71 VLPs to induce the production of IL-12 p40, IL-12 p70, and IL-10 by DCs and inhibited EV71 VLPs binding to DCs. Our study findings clarified the important role for TLR4 signaling in DCs in response to EV71 VLPs and showed that EV71 VLPs induced inhibitor of kappaB alpha (IκBα) degradation and nuclear factor of kappaB (NF-κB) activation.

Hepatitis B virus Ag (HBsAg), a major antigen of hepatitis B virus (HBV), is also a vaccine component for prevention of HBV infection. Dendritic cells (DCs) of HBV carriers reportedly exhibit functional impairment. In this study, the aim was to investigate the effect of HBsAg on activation of human monocyte-derived dendritic cells (MD-DCs), and the subsequent signal transduction pathway. Treatment of MD-DCs with HBsAg resulted in enhanced cell surface expression of cluster of differentiation 80, CD83, CD86 and major histocompatibility complex class II, and increased interleukin (IL)-12 p40, IL-12p70, and IL-10 production. Furthermore, HBsAg treatment of MD-DCs with HBsAg resulted in enhanced T cell-stimulatory capacity and increased T cell secretion of interferon and IL-10. The pathway of MD-DCs activation by HBsAg was further investigated in the present study. Inhibition of nuclear factor (NF)-kappa B (κB) by helenalin and p38 mitogen-activated protein kinase (MAPK) by SB203580 prevented production of IL-12 p40, IL-12 p70, and IL-10. HBsAg also augmented MAPK phosphorylation. Thus, cytokine secretion of human MD-DCs by HBsAg is blocked by inhibition of the NF-κB and p38 MAPK pathways. Likewise, decreased inhibition of kappa B alpha concentrations and MAPK phosphorylation are critical for MD-DC maturation by HBsAg. These findings may provide a strategy for improving the prophylactic and therapeutic efficacy of vaccines and tumor therapies that utilize these pathways., (© 2012 The Societies and Wiley Publishing Asia Pty Ltd.)

Although chronic hepatitis C patients have a lower frequency and functions of dendritic cells (DCs) than healthy subjects, little is known about the serial changes in frequency and functions of DCs following anti-viral treatment and the relationship with treatment outcomes. Twenty patients with hepatitis C virus genotype 1 receiving peginterferon (PEG-IFN) and ribavirin for 24 weeks were enrolled. The frequency and functions of DCs were assayed at baseline and 24 weeks post-treatment. Ten sex and age-matched healthy adults served as controls. Nineteen of the 20 chronic hepatitis C patients completed 24 weeks of combination therapy. Fifteen patients achieved rapid virologic response and 12 achieved sustained virologic response (SVR). The baseline frequency of peripheral blood myeloid DCs and plasmacytoid DCs was significantly lower in chronic hepatitis C patients than in healthy controls. In patients who achieved SVR, the frequency of DCs subsets at the end of follow-up increased to a level comparable to healthy controls. Although no functional defects of DCs was found in chronic hepatitis C patients in comparison with healthy controls, in patients without SVR had a lower CD83 expression and higher interleukin-10 production of DCs than SVR patients. The results suggest that low CD83 expression and high IL-10 production of DCs at the baseline may predict a poor virologic response to 24-week PEG-IFN plus ribavirin therapy in HCV genotype 1 patients., (Copyright © 2011 Wiley-Liss, Inc.)

Background: Ganoderma lucidum-derived polysaccharide (PS-G) can rapidly and effectively promote the activation and maturation of immature dendritic cells (DCs), suggesting that PS-G possesses the capacity to regulate immune responses. This study aimed to clarify the immunologic effect of PS-G on monocyte-derived dendritic cells (MD-DCs) from asthmatic children allergic to house dust mites. The MD-DCs were stimulated for 24 h with the related allergen, Der p 1, in the presence or absence of PS-G. Cell surface markers and phagocytic capacity were assessed by FACS analysis, and key polarizing cytokines (IL-12 p40, IL-12 p70, IL-6, IL-23, and IL-10) were quantified. The subsequent regulatory effect of pulsed MD-DCs on naïve T cells was evaluated by determining the T-cell cytokine profile., Results: PS-G induced the maturation of MD-DCs and decreased phagocytic capacity, even if pulsed with Der p 1. After incubation with PS-G and Der p 1, MD-DCs produced higher amounts of IL-12 p70, IL-12 p40, IL-6, IL-23, and IL10 than Der p 1-pulsed DCs. Furthermore, type 1 helper T (Th1) cell cytokine (INF-γ) production was highly increased when naïve autologous T cells were co-cultured with Der p 1-pulsed MD-DCs. Naïve T cells stimulated by MD-DCs pulsed with Der p 1 failed to produce proliferation of T-cells, whereas the addition of PS-G to Der p 1 induced a significant proliferation of T-cells similar to that observed with PS-G alone., Conclusion: The presence of PS-G in an allergen pulse promoted allergic MD-DCs to produce IL-12 p70, IL-12 p40, IL-6, IL-23, and IL-10, and exerted an effect on shifting the immune balance towards Th1 in children with allergic asthma.

Hepatitis B virus surface antigen (HBsAg) is a major antigen of hepatitis B virus (HBV). Dendritic cells (DC) of HBV carriers have been reported to exhibit functional impairment. In this study, the role of HBsAg on mice bone marrow-derived dendritic cells and immune responses in vivo was studied. The immune modulatory function of HBsAg was explored by using mice bone marrow-derived dendritic cells in vitro and also by examining an ovalbumin (OVA) specific immune response in vivo. Treatment of dendritic cells with HBsAg resulted in enhanced cell surface expression of cluster of differentiation (CD) 80, CD83, CD86, and major histocompatibility complex (MHC) class II, and enhanced production of interleukin (IL)-12 p40 and IL-12 p70. Treatment of dendritic cells with HBsAg resulted in decreased T cell secretion of IL-5 by OVA stimulation. In addition, the results showed stronger OVA-specific immunoglobulin (Ig) M and weaker IgG responses in mice sera when they had been immunized with OVA and co-injected with HBsAg. It was also found that the mice exhibited significant enhancement of anti-OVA IgG2a antibody (Ab), as well as marked inhibition of IgG1 Ab production. In cellular immune responses, IL-5 production was significantly decreased and interferon (IFN)-γ increased in the group co-injected with HBsAg. On the other hand, the induction of lymphoproliferative response to OVA stimulation in spleen cells was decreased in the HBsAg co-injected group. These results demonstrate that HBsAg can affect the differentiation of T helper (Th) cells, which might provide a strategy for improving its prophylactic and therapeutic efficacy., (© 2010 The Societies and Blackwell Publishing Asia Pty Ltd.)

An autoimmune response can be induced with the application of dendritic cells (DCs), offering a viable tumor vaccine for cancer immunotherapy. Previous studies have shown that DC-based tumor vaccines in animal tumor models can inhibit tumor growth and induce autoantibodies transiently without clinical or histological features of autoimmunity. The present study aimed to investigate the role of immune response induced by dendritic cells-based therapy, especially anti-ds DNA antibodies in tumor inhibition. In this study, high titers of anti-ds DNA antibodies were induced after injecting syngeneic dendritic cells into BALB/c mice. In addition, mice immunized with DCs showed the inhibition of RL male symbol 1, BALB/c leukemia cell line, tumor growth, and prolonged survival times of tumor mice but no significant difference was found in specific CTL response and NK cell activity when compared to those of the control group. Anti-ds DNA monoclonal antibodies can recognize RL male symbol 1 cells but not normal cells by FACS analysis. Monoclonal anti-ds DNA antibody was demonstrated to be able to lyse tumor cells via complement mediated reaction in vitro and also exhibits the anti-tumor effects when the antibody was injected into tumor-implanted mice. The data suggested that immunization with dendritic cells can induce autoimmune response, which might exert anti-tumor activity in vivo.

Ganoderma lucidum, an oriental medicinal mushroom, has been widely used in Asia to promote health and longevity. LZ-8 is a protein derived from the fungus G. lucidum and has immunomodulatory capacities. In this study, we investigated the immune modulatory effects of rLZ-8 on human monocyte-derived DCs. Treatment of DC with rLZ-8 resulted in the enhanced cell-surface expression of CD80, CD86, CD83, and HLA-DR, as well as the enhanced production of IL-12 p40, IL-10, and IL-23, and the capacity for endocytosis was suppressed in DCs. In addition, treatment of DCs with rLZ-8 resulted in an enhanced, naïve T cell-stimulatory capacity and increased, naïve T cell secretion of IFN-gamma and IL-10. Neutralization with antibodies against TLR4 inhibited the rLZ-8-induced production of IL-12 p40 and IL-10 in DCs. rLZ-8 can stimulate TLR4 or TLR4/MD2-transfected HEK293 cells to produce IL-8. These results suggested an important role for TLR4 in signaling DCs upon incubation with rLZ-8. Further study showed that rLZ-8 was able to augment IKK, NF-kappaB activity, and also IkappaBalpha and MAPK phosphorylation. Further, inhibition of NF-kappaB by helenalin prevented the effects of rLZ-8 in the expression of CD80, CD86, CD83, and HLA-DR and production of IL-12 p40 and IL-10 in various degrees. To confirm the in vitro data, we investigated the effect of rLZ-8 further on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with OVA/rLZ-8 showed that the anti-OVA IgG2a, IFN-gamma, and IL-2 were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our experiments demonstrated that rLZ-8 can effectively promote the activation and maturation of immature DCs, preferring a Th1 response, suggesting that rLZ-8 may possess a potential effect in regulating immune responses.

Residual cancer cells appearing in blood circulation reduce the effects of radiotherapy or chemotherapy in cancer patients. It has been well documented that cultured dendritic cells can be used as a powerful tool to induce immune response. In this study, we administered different manipulations of dendritic cells (DCs), including DCs pulsed with tumor cell lysate (TCL), transfected with adenoviral IL-12 vector (AdIL-12) and transfected with AdIL-12 after being pulsed with TCL, to determine whether improved DCs based immunotherapy can specifically suppress the metastasis of tumor cells. The results demonstrated that administration of engineered DCs that transfected with AdIL-12 after being pulsed with TCL to mice with leukemia had a better protective effect than that of DCs either pulsed with TCL or transfected with AdIL-12. Moreover, depletion of CD8(+) cells in the engineered DCs administered leukemia mice reduced the protective effect. These results suggest that DCs modified with TCL and AdIL-12 can prolong survival time by enhancing the activity of cytotoxic T cells. Although more studies on the mechanisms are needed, cytokine genes engineered DCs provide a promising therapeutic potential on the murine model of leukemia.

Background: Propolis, an ancient herbal medicine, has been reported the beneficial effect both in asthma patients and murine model of asthma, but the mechanism was not clearly understood. In this study, the effect of caffeic acid phenethyl ester (CAPE), the most extensively studied components in propolis, on the functions of human monocyte-derived dendritic cells (MoDCs) was investigated., Results: CAPE significantly inhibited IL-12 p40, IL-12 p70, IL-10 protein expression in mature healthy human MoDCs stimulated by lipopolysaccharides (LPS) and IL-12 p40, IL-10, IP-10 stimulated by crude mite extract. CAPE significantly inhibited IL-10 and IP-10 but not IL-12 expression in allergic patients' MoDCs stimulated by crude mite extract. In contrast, the upregulation of costimulatory molecules in mature MoDCs was not suppressed by CAPE. Further, the antigen presenting ability of DCs was not inhibited by CAPE. CAPE inhibited IkappaBalpha phosphorylation and NF-kappaB activation but not mitogen-activated protein kinase (MAPK) family phosphorylation in human MoDCs., Conclusion: These results indicated that CAPE inhibited cytokine and chemokine production by MoDCs which might be related to the NF-kappaB signaling pathway. This study provided a new insight into the mechanism of CAPE in immune response and the rationale for propolis in the treatment of asthma and other allergic disorders.

Placental growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family and represents a key regulator of angiogenic events in development and pathologic conditions. In this study, PlGF-modulated differentiation and maturation of human dendritic cells (DCs) from CD14+ monocytes were investigated. The DC, differentiated from CD14+ monocytes in the presence of PlGF during 5 days, was referred to as "PlGF-DC", in contrast to the "classical-DC", obtained in the absence of PlGF. Treatment of PlGF-DC or classical-DC with PlGF resulted in the down-regulation of CD80, CD86, CD83, CD40, and HLA-DR expression, and CD1a was increased, as well as the inhibition of IL-12 p70, p40, IL-8, and TNF-alpha production in response to LPS stimulation. This PlGF-induced DC dysfunction was recovered by anti-human VEGF receptor 1 mAb. In addition, treatment of PlGF-DC or classical-DC with PlGF resulted in the suppression of naïve CD4+ T cell proliferation in an allogenic MLR but up-regulated the IL-5 and IL-13 secretion of the CD4+ T cell. PlGF was also able to inhibit LPS-induced IkappaBalpha phosphorylation and NF-kappaB activity. Taken together, our data demonstrate that the immunosuppressive properties of PlGF are through the NF-kappaB signaling pathway. PlGF might play a major role in the pathogenesis of tumors and act as an effector molecule to skew T cell response to the Th2 phenotype, which might be more beneficial for pregnancy.

Ganoderma lucidum is a medicinal mushroom in China and other Asian countries. The polysaccharide from G. lucidum (PS-G) is a branched (1-->6)-beta-d-glucan moiety. In this study, we examined the effects of PS-G on human monocyte-derived dendritic cells (DCs) with microarray analysis by Human Genome U133 Plus 2.0 GeneChip. In comparing mean signal values between PS-G-treated DCs with untreated DCs, 3477 (17%) probe sets were up-regulated, and 4418 (19%) probe sets were down-regulated after PS-G treatment. These results demonstrate that genes associated with phagocytosis (CD36, CD206, and CD209) are decreased and genes associated with proinflammatory chemokines (CCL20, CCL5, and CCL19), cytokines [interleukin (IL)-27, IL-23A, IL-12A, and IL-12B], and costimulatory molecules (CD40, CD54, CD80, and CD86) are increased. To confirm the microarray data, we further investigated the effect of PS-G on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with ovalbumin (OVA)/PS-G showed that the anti-OVA IgG2a levels were significantly increased compared with OVA alone in BALB/c mice. Together, our data demonstrate that PS-G could effectively promote the activation and maturation of immature DCs, preferring a T helper 1 response. Furthermore, the results also demonstrate that the data from microarray analysis could be correlated with the in vivo effect of the immune-enhancing compound.

Ganoderma lucidum, a fungus native to China, has been widely used to promote health and longevity in the Chinese. The polysaccharide component with a branched (1-->6)-beta-D-glucan moiety of G. lucidum (PS-G) has been reported to exert anti-tumor activity and activation of natural killer cells. In this study, we investigated the effects of PS-G on human monocyte-derived dendritic cells (DC). Treatment of DC with PS-G resulted in the enhanced cell-surface expression of CD80, CD86, CD83, CD40, CD54, and human leukocyte antigen (HLA)-DR, as well as the enhanced production of interleukin (IL)-12p70, p40, and IL-10 and also IL-12p35, p40, and IL-10 mRNA expression, and the capacity for endocytosis was suppressed in DC. In addition, treatment of DC with PS-G resulted in enhanced T cell-stimulatory capacity and increased T cell secretion of interferon-gamma and IL-10. Neutralization with antibodies against Toll-like receptor (TLR)-4 inhibited the PS-G-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-4 in signaling DC upon incubation with PS-G. Further study showed that PS-G was able to augment inhibitor of kappaB (IkappaB) kinase and nuclear factor (NF)-kappaB activity and also IkappaB alpha and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Further, inhibition of NF-kappaB by helenalin and p38 MAPK by SB98059 prevented the effects of PS-G in the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR and production of IL-12p70, p40, and IL-10 in various degrees. Taken together, our data demonstrate that PS-G can effectively promote the activation and maturation of immature DC, suggesting that PS-G may possess a potential in regulating immune responses.