Paeoniflorin suppresses IL-6/Stat3 pathway via upregulation of Socs3 in dendritic cells in response to 1-chloro-2,4-dinitrobenze

Mounting evidence has suggested that inflammation is associated with IL-6/Stat3 pathway in dendritic cells (DCs) and Th17 cells, which are critical for development of allergic contact dermatitis (ACD). Paeoniflorin (PF) has been clinically proved to be effective in the treatment of inflammatory skin diseases such as ACD. We have previously demonstrated the effect of PF on DCs stimulated with 1-chloro-2,4-dinitrobenze (DNCB) and naïve CD4⁺ CD45RA⁺ T cells for Th17 cell differentiation. However, whether PF down-regulates IL-6/Stat3 in DCs and Th17 cells remains to be explored. In this study, we show clearly that PF markedly decreases IL-6/Stat3 in DCs stimulated with DNCB at both gene and protein levels compared with control DCs in vitro. Meanwhile, PF up-regulates suppressor of cytokine signaling 3 (Socs3). Such decreased expression of IL-6/Stat3 is abolished in DCs that were transfected with Socs3 short interfering RNA (siRNA). When mice CD4⁺ CD45 RA⁺ T cells were co-cultured with PF-treated DCs stimulated with/without DNCB, the gene expression of the Th17 cell markers such as retinoic acid-related orphan nuclear hormone receptor γt (RORγt), IL-17A, and IL-23R decreased, in accordance with the less secretions of IL-17 and IL-23 in vitro and in vivo. Finally, the suppressed Th17 differentiation induced by PF can be abolished by additional recombinant mouse IL-6. Our results suggest that the anti-inflammatory mechanisms introduced by depletion of Socs3 expression or inactivation of the negative regulator such as Socs3 may represent a promising strategy for the prevention of ACD.     

Effective stimulation of invariant natural killer T cells by oligomannose-coated liposomes

vThe in vitro and in vivo response of invariant natural killer T (iNKT) cells to alpha-galactosylceramide (αGC)-containing oligomannose-coated liposomes (αGC-OMLs) was examined to determine whether selective delivery of αGC to dendritic cells (DCs) and subsequent activation of iNKT cells could be achieved. Splenocytes stimulated with αGC-OMLs produced higher levels of IFN-γ compared to those stimulated with bare liposomes without an oligomannose coating (αGC-BLs). The ratio of IFN-γ/IL-4 produced from αGC-OML-treated splenocytes was higher than those produced from αGC-BL- and soluble αGC-treated cells. Depletion of CD3⁺-, DX5⁺- or CD11c⁺-cells from splenocytes almost completely abolished the αGC-OML-stimulated cytokine production, suggesting that both NKT cells and DCs were involved in the response to αGC-OML stimulation. In addition, αGC-OMLs were incorporated into both splenic and bone marrow-derived DCs more effectively than αGC-BLs. iNKT cells stimulated with DCs with ingested αGC-OMLs produced much higher levels of IFN-γ than those stimulated with DCs containing αGC-BLs or soluble αGC. Systemic administration of αGC-OMLs led to modification of the kinetics of IFN-γ production in vivo and also resulted in predominant production of IFN-γ from splenocytes over IL-4. In addition, iNKT cells proliferated and expanded upon in vivo activation of the cells with αGC-OMLs much more extensively than with αGC-BLs or soluble αGC. Collectively, our results suggest that αGC-OMLs can be used as a preferential delivery system for lipid antigens to DCs to activate iNKT cells in vivo and ex vivo.     

Thymol as a reciprocal regulator of T cell differentiation: Promotion of regulatory T cells and suppression of Th1/Th17 cells

Regulatory T cells (Tregs) are critical for maintaining immune response and enhancing their differentiation has therapeutic implications for autoimmune diseases. In this study, we investigated the effects of thymol a well-known monoterpene from Thyme on differentiation and function of Tregs. In vitro generation of Tregs from purified naïve CD4⁺CD25⁻ T cells in the presence of thymol was carried out. Suppressor activity of generated Tregs was examined by changes in the proliferation of CFSE-labeled conventional T cells. Thymol promotes differentiation of naïve CD4⁺CD25⁻ T cells to CD4⁺CD25⁺Foxp3⁺ Tregs [66.9–71.8% vs. control (47%)] and increased intensity of Foxp3 expression on Tregs (p < 0.01). In functional assay, an increased immune suppression by thymol-induced Tregs (≈2.5 times of untreated Tregs) was detected. For in vivo study, thymol was intraperitoneally administered to ovalbumin (Ova)-immunized mice. Flow cytometry assessment of spleens from thymol-treated Ova-immunized mice showed increased number of CD4⁺ Foxp3⁺ Tregs (>8%, p < 0.01(and decreased levels of CD4⁺T-bet⁺ Th1 and CD4⁺RORγt⁺ Th17 cells resulted in significant decreased Th1/Treg and Th17/Treg ratios. In ex vivo Ova challenge of splenocytes from thymol-treated Ova-immunized mice, similarly higher levels of CD4⁺ Foxp3⁺ Tregs, and also elevated TGF-β expression in CD4⁺Foxp3⁺ population (48.1% vs. 18.9% in untreated Ova-immunized group) and reduced IFN-γ-producing CD4⁺T-bet⁺ T cells and IL-17-producing CD4⁺RORγt⁺ T cells were detected. This led to marked decreased ratios of IFNγ/TGF-β and IL-17/TGF-β expressions. In conclusion, this study revealed thymol as a compound with enhancing effects on Treg differentiation and function, which may have potential benefits in treatment of immune-mediated diseases with Th1/Th17 over-activation.     

Maturation of dendritic cells by maitake α-glucan enhances anti-cancer effect of dendritic cell vaccination

Dendritic cells (DCs) play a primary role in antigen presentation to CD4⁺ and CD8⁺ T cells and induce acquired immune response against cancer cells. Therefore, determining positive modulators of DC activation to improve therapeutic approaches for cancer immunotherapy is greatly needed. In this study, we investigated the effect of maitake α-glucan YM-2A, isolated from Grifola frondosa, on the maturation and function of DCs and its adjuvant effect on a tumor-associated antigen (TAA)-loaded DC vaccine against murine tumor. We showed that YM-2A induced morphological changes and increased cell-surface maturation markers and cytokine production in DCs. In a mixed lymphocyte reactions assay, YM-2A-treated DCs increased proliferation and production of IFN-γ by allogeneic CD4⁺ and CD8⁺ T cells. These results indicate that YM-2A phenotypically and functionally activates DCs. Furthermore, YM-2A-treated TAA-loaded DC vaccine significantly reduced tumor growth and improved survival in two murine tumor models, CT-26 tumor-bearing BALB/c mice and B16 melanoma-bearing C57BL/6 mice. YM-2A-treated TAA-loaded DC vaccine increased splenic IFN-γ producing CD4⁺ and CD8⁺ T cells in CT-26 tumor-bearing BALB/c mice. Antibody neutralization studies indicated that YM-2A-induced DC maturation is mediated, in part, by the Dectin-1-dependent pathway. Overall, YM-2A-treatment with a TAA-loaded DC vaccine could be an excellent candidate for immunotherapy against cancer.     

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.     

CD4+ B220+ TCRγδ+ T cells produce IL-17 in lupus-prone MRL/lpr mice

Systemic lupus erythematosus is an autoimmune disease with comprehensive immune cell disorders. Recent studies suggested that pro-inflammatory cytokine IL-17 plays important role in lupus, leaving the cellular sources and their pathogenic and physiologic characters largely unknown. In the current study, by using lupus-prone MRL/lpr mice, we demonstrated that Th17 response prevails in lupus disease regarding significantly accumulated serum IL-17, increased IL-17-producing splenocytes, and elevated phospho-STAT3 in CD4⁺ T cells. Intracellular staining revealed that unusual CD4⁺ B220⁺ T cells are major IL-17-producing cells, whereas conventional CD4⁺ B220⁻ T cells are major IFN-γ-producing cells. Subsequent studies showed that CD4⁺ B220⁺ cells contains both αβ and γδ T cells in the spleen and thymus of MRL/lpr mice. Further study showed that around 60% of γδ T cells in MRL/lpr mice co-express both B220 and CD4 on their surface, and are the major RORγt⁺ cells in MRL/lpr mice. Finally, CD4⁺ B220⁺ T cells alone do not proliferate, but could enhance the proliferation and IFN-γ-production of conventional CD4⁺ B220⁻ T cells. Our findings suggest the pathogenic role of unusual CD4⁺ B220⁺ T cells in lupus disease in MRL/lpr mice according to their IL-17-producing ability and stimulatory function for conventional CD4⁺ B220⁻ T cells.     

The shiitake mushroom-derived immuno-stimulant lentinan protects against murine malaria blood-stage infection by evoking adaptive immune-responses

Lentinan, a (1-3)-beta glucan from Lentinus edodes, is an effective immunostimulatory drug. We tested the effects of lentinan during blood-stage infection by Plasmodium yoelii 17XL (P.y17XL). Pre-treatment of mice with lentinan significantly decreased the parasitemia and increased their survival after infection. Enhanced IL-12, IFN-γ and NO production induced by lentinan in spleen cells of infected mice revealed that the Th1 immune response was stimulated against malaria infection. In vitro and in vivo, lentinan can result in enhanced expression of MHC II, CD80/CD86, and Toll-like receptors (TLR2/TLR4), and increased production of IL-12 in spleen dendritic cells (DCs) co-cultured with parasitized red blood cells (pRBCs). Moreover, both the number of CD4⁺CD25⁺ regulatory T cells (Tregs) and the levels of IL-10 secreted by Tregs were reduced by pre-treatment with lentinan in the spleen of malaria-infected mice. Meanwhile, apoptosis of CD4⁺ T cell in spleens of mice pretreated with lentinan was significantly reduced. In summary, lentinan can induce protective Th1 immune responses to control the proliferation of malaria parasites during the blood-stage of P.y17XL infection by stimulating maturation of DCs to inhibit negative regulation of the Th1 immune response by Tregs. Taken together, our findings suggest that lentinan has prophylactic potential for the treatment of malaria.     

The source of Mycobacterium tuberculosis-specific IFN-γ production in peripheral blood mononuclear cells of TB patients

Mycobacterium tuberculosis (Mtb)-specific IFN-γ secretion plays important roles in anti-tuberculosis (TB) immunity. Mtb-specific IFN-γ response can be induced in HIV/TB co-infected patients with a low CD4 lymphocyte count; this suggests that the source of Mtb-specific IFN-γ production is not limited in CD4⁺ T lymphocytes. Currently, the major sources of Mtb-specific IFN-γ production and the function and phenotype of Mtb-specific IFN-γ-producing cells still remain unclear. Thirty-nine participants (24 active TB patients, 10 HIV/TB co-infected patients, and 5 healthy volunteers) were recruited according to conventional tests and Mtb-specific IFN-γ ELISPOT assay. Multicolor flow cytometry was used to investigate the production of intracellular IFN-γ in peripheral blood mononuclear cells (PBMCs) after Mtb-specific antigen stimulation. Our results showed that CD4⁺, CD8⁺ T cells and NK cells are all major sources of Mtb-specific IFN-γ production in PBMCs of TB patients. Moreover, CD8⁺ T cells are the highest number of Mtb-specific IFN-γ-producing cells in HIV/TB co-infected patients. Although the activity of NK cells is significantly reduced in TB patients when compared with healthy controls, Mtb-specific antigen stimulation induces a significant increase in NK cell activity. We also showed that CD45RO is the characteristic marker of Mtb-specific IFN-γ-producing T cells but not that of Mtb-specific IFN-γ-producing NK cells in peripheral blood. High expression of CD11a may be the characteristic feature of Mtb-specific IFN-γ-producing NK cells. This study put forward a new insight on the source of antigen-specific IFN-γ-production in PBMCs of TB patients.     

In vitro induced CD4+CD25+Foxp3+ Tregs attenuate hepatic ischemia–reperfusion injury

Reperfusion injury causes liver dysfunction after warm or cold ischemia. Emerging data suggest a role of T cells as mediators in this ischemia/reperfusion (I/R) injury. In the T cells, a part of CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) were reported to facilitate recovery from I/R injury. These Tregs can be induced by TGF-β in vitro. Interestingly, rapamycin was reported to selectively expand these Tregs in vitro. In the present study, addition of rapamycin to cultures containing TGF-β further increased the frequency and absolute number of functional CD4⁺ Tregs. Using a partial (70%) hepatic warm ischemia model, we investigated the effects of liver function recovery under the treatment of Tregs induced by rapamycin and TGF-β. The treatment of Tregs significantly reduced serum alanine aminotransferase and aspartate aminotransferase compared to I/R control animals at 24 h after reperfusion (P < 0.05). They also significantly attenuated the up-regulation of IFN-γ and IL-17 compared to the I/R control animals (P < 0.05). In conclusion, Tregs ameliorate the biochemical of hepatic I/R injury by preventing proinflammatory cytokines following a warm I/R insult. These data may pave the way to use Tregs as cell therapy to prevent hepatic I/R injury.     

Induction of dendritic cell maturation by β-glucan isolated from Sparassis crispa

Sparassis crispa is a medicinal mushroom containing high 6-branched 1,3-β-D-glucan (sparan) content, which exhibits immune-mediated antitumor activity. In the present study, we investigated the stimulating effect of sparan on phenotypic and functional maturation of dendritic cells (DCs). Phenotypic maturation was confirmed by the elevated expressions of CD40, CD80, CD86, and MHC-I/II molecules. Functional activation was proved by increased cytokine production of IL-12, IL-1β, TNF-α, and IFN-α/β, enhanced IL-2 production and proliferation of allogenic T cells, and decreased endocytosis. The role of toll-like receptor 4 (TLR4) as a membrane receptor of sparan was proved by the impaired maturation of DCs generated from bone marrow cells of tlr4^−/− knock-out mice and TLR4-mutated C3H/HeJ mice, and by using anti-MD-2/TLR4 neutralizing antibody. Sparan increased phosphorylation of ERK, p38, and JNK, and enhanced nuclear translocation of NF-κB p50/p65 in DCs. These results indicate that sparan activates DCs via MAPK and NF-κB signaling pathways, which are signaling molecules downstream of TLR4.     

Dendritic cells transfected with indoleamine 2,3-dioxygenase gene suppressed acute rejection of cardiac allograft

BackgroundImmunomodulation by indoleamine 2,3-dioxygenase (IDO) has been documented in many studies yet its underlying mechanisms remain undefined, especially in solid organ transplantation. Recent research demonstrated that the active expression of IDO in dendritic cells (DCs) regulates immune reaction. This study assessed whether DCs transfected with IDO gene inhibit T cells responses and suppress cardiac allograft rejection.MethodsAdenovirus vector containing IDO gene was transfected into DCs to obtain IDO-positive DCs (IDO⁺ DCs). To evaluate the effect of IDO⁺ DCs on T cells in vitro, CD4⁺ T cell proliferation and apoptosis was assessed in mixed lymphocyte reactions and measured by flow cytometry, respectively. IDO⁺ DCs from C57BL/6 mice were injected into BALB/c recipients before heterotopic cardiac transplantation.ResultsSupernatant fluids from cultures of IDO⁺ DCs had decreased tryptophan and increased kynurenine levels, reflecting IDO activity. IDO⁺ DCs suppressed CD4⁺ T cell responses in vitro, as reflected by decreased proliferation and increased apoptosis. In the transplant model, IDO⁺ DCs prolonged survival and alleviated rejection of cardiac allograft in recipients injected with IDO⁺ DCs. In vivo, IDO⁺ DCs also significantly impaired CD4⁺ T cell responses promoting increased apoptosis and a Th2-dominant cytokine shift.ConclusionsIDO overexpression in DCs suppressed T cells alloresponses in vitro, and IDO⁺ DCs attenuated acute allograft rejection in vivo. Regulation of tryptophan catabolism by means of IDO overexpression in DCs may be a useful approach in cardiac transplantation and immunological tolerance.     

Astragalus polysaccharides enhance immune responses of HBV DNA vaccination via promoting the dendritic cell maturation and suppressing Treg frequency in mice

Astragalus polysaccharides (APS), an extract from a kind of Chinese traditional herb Astragalus membranaceus, was proved to have strong immunoregulatory properties. In this study, APS was employed as an adjuvant of Hepatitis B virus (HBV) DNA vaccine (pcDS2) and its' effects on immune system of mice were investigated. Our data demonstrated that APS as an adjuvant could increase the HBsAg-specific antibody level as well as the proliferating activity of T cells. APS also could induce CD4⁺ T cells to produce IL-4, IL-2 and IFN-γ and enhance IFN-γ expression of CD8⁺ T cells. Moreover, APS could induce the robust activity of the cytotoxic lymphocytes (CTL). Additionally, APS could stimulate the dendritic cells (DC) maturation which is characterized by up-regulation of MHC I/II, CD40, CD80 and CD86, and decreased the frequency of the regulatory T cells (nTreg). Collectively, these findings suggest that APS is a potent adjuvant for the hepatitis B DNA vaccine and can enhance the immune responses of HBV DNA vaccine via promoting DC maturation and inhibit the Treg frequency.     

Triptolide regulates T cell-mediated immunity via induction of CD11c dendritic cell differentiation

Triptolide(TPT) isolated from one of the Chinese herbs, Tripterygium wilfordii Hook. F. (TWHF), are known to have a variety of immunomodulatory activities. This study was performed to investigate the effect of TPT on the differentiation of splenic DCs and its influence on T cell-mediated immunity regarding to DC subsets CD11c^lowI-a/e^lowCD45RB⁺(CD11c^low DCs) and CD11c^highI-a/e^highCD45RB^- (CD11c^high DCs) in male C57BL/6 mice spleens in vitro. The percentage of CD11c^low DCs was significantly increased after treatment with TPT compared to their counterparts (CD11c^high DCs). It was found that unlike the gradually decreasing interleukin (IL)-12 secretion of CD11c^high DCs induced by TPT, CD11c^low DCs showed a obvious dose-dependent response between the increasing of IL-10 production and TPT stimulation. After treatment with anti-IL-12R or anti-IL-10 monoclonal antibody in CD4⁺ T cells + CD11c^high DCs or CD11c^low DCs mixed lymphocyte reaction, the induction of these DCs on T cells was inhibited dramatically. These data demonstrated that TPT might induce the differentiation of splenic DCs to CD11c^low DCs followed by shifting of Th1 to Th2 with enhancement of T lymphocyte immune function in vitro.     

Levamisole promotes murine bone marrow derived dendritic cell activation and drives Th1 immune response in vitro and in vivo

Our lab previously found that levamisole (LMS) as an adjuvant enhanced the efficacy of vaccine against infectious pathogens. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that BALB/c bone marrow-derived DC stimulated with LMS resulted in enhanced cell-surface expression of CD80, CD86, CD40 and MHC class II, as well as enhanced production of IL-12p70, TNF-α and IL-1β. Interestingly, the LMS activated DCs were able to stimulate CD4⁺ T cell proliferation and facilitated Th1 differentiation by increasing the secretion of IFN-γ in an allogeneic mixed leukocyte reaction. Furthermore, to confirm the in vitro data, we investigated the effect of LMS on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with LMS plus OVA showed that anti-OVA IgG2a and IFN-γ were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our results suggested that murine bone marrow-derived DC, played a crucial role in the effect of LMS on the induction of Th1 responses, which probably was due to its ability to promote DC maturation and secrete proinflammatory cytokines.     

Polyactin A is a novel and potent immunological adjuvant for peptide-based cancer vaccine

Synthetic peptide cancer vaccines are poorly immunogenic sub-unit vaccines and thus essentially need adjuvants in their formulations to increase the efficacy by enhancing the peptide-specific immune response. However, aluminum-based compounds are almost dependent for clinical use at present. Therefore, the increasing use of peptide-based vaccines makes the need for novel and potent adjuvants. Polyactin A (PAA) has been used for the clinical treatment of impaired immunity in China for over 30 years. To figure out the adjuvant effects of PAA for E75 peptide breast cancer vaccine (Her2 p369-p377), the generation of mature dendritic cells (DCs) from peripheral blood monocytes (PBMCs) cultured with PAA, IL-4 and TNF-α was assessed by morphologic features and the expressions of special surface markers using flow cytometry. Then the functional features of PBMCs-derived DCs cultured with PAA, IL-4 and TNF-α were investigated by inducing E75-specific cytotoxicity. Finally, C57BL/6-Tg(HLA-A2.1)1Enge/J transgenic mice were immunized with E75 and various amounts of PAA, and splenic lymphocyte proliferation and the IFN-γ level were determined. The results showed that PAA, just like granulocyte-macrophage colony stimulating factor, with IL-4 and TNF-α efficiently induced mature DCs from PBMCs, and these DCs could trigger a potent E75 peptide-specific CD8⁺ T-cell response in vitro. Immunization with E75 and PAA significantly increased positive rates of CD4⁺ and CD8⁺ T lymphocytes, and enhanced splenocytes proliferation and levels of IFN-γ in splenocytes when induced by E75. Our results indicated that PAA could efficiently induce E75-specific immunologic responses in vitro and in vivo. Therefore, PAA possesses potent adjuvant effect on peptide-based cancer vaccine. Our study provides a safe, effective and novel adjuvant for clinical use.     

Upregulation of PD-1 on CD4+CD25+T cells is associated with immunosuppression in liver of mice infected with Echinococcus multilocularis

Alveolar echinococcosis is a zoonotic disease caused by Echinococcus multilocularis (E. multilocularis) infection. The relationship between PD-1/PD-L1 pathway and Tregs at different stages of E. multilocularis infection has rarely been reported. This study aims to investigate the role of PD-1/PD-L1 in immunosuppression of Tregs in E. multilocularis infection. Hematoxylin-eosin staining, flow cytometry, immunohistochemistry, quantitative RT-PCR analysis, cytometric bead array and MTT assay were used to analyze liver pathological changes, percentages of PD-1⁺ Tregs and PD-L1⁺ dendritic cells (DCs), expression levels of PD-1, PD-L1 and Foxp3, levels of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) and proliferation of lymphocytes. During middle-late stage (day 30 to day 330) the percentages of PD-1⁺ Tregs and PD-L1⁺ DCs together with levels of Foxp3, IL-10 and TGF-β increased significantly and maintained at high level. The expression of PD-1 and PD-L1 was increased with the enlarging erosion of E. multilocularis, and was mainly distributed in hepatic sinus, fibrous wall of alveolar hydatid and germinal layer around foci of infection. PD-1/PD-L1 promoted the secretion of IL-10 and TGF-β. Our results indicate that engagement of the PD-1 and PD-L1 correlates with inhibition of T-cell effector function, cytokine secretion and proliferation. High expression of PD-1/PD-L1 may play an important role in stimulating CD4⁺CD25⁺ T cells, and maintaining peripheral tolerance and immune evasion during chronic infection of E. multilocularis.     

Loxoribine,a selective Toll-like receptor 7 agonist,induces maturation of human monocyte-derived dendritic cells and stimulates their Th-1- and Th-17-polarizing capability

Recently, a guanosine analog, 7-allyl-7,8-dihydro-8-oxo-guanosine (loxoribine), has been identified as a selective Toll-like receptor (TLR)7 agonist. Bearing in mind the controversy regarding the expression of TLR7 by human myeloid dendritic cells (DCs) and its significance for functions of these cells, the goal of this study was to investigate the effect of loxoribine on differentiation, maturation and functions of human monocyte-derived (Mo)DCs. Immature MoDCs were obtained by cultivation of monocytes for 6 days with recombinant granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. These cells were stimulated with loxoribine (250 μM) for an additional 48 h. Phenotypic properties of MoDCs were determined by flow cytometry, cytokine production was assayed by ELISA, whereas their allostimulatory capability was tested using a mixed leukocyte reaction. We showed that loxoribine up-regulated the expression of TLR7, CD40, CD54, CD80, CD83 and CCR7 and stimulated the production of IL-12, IL-23, IL-27 and IL-10 by MoDCs, whereas the level of interferon (IFN)-β was not modulated. Allogeneic CD4⁺T cells in co-culture with loxoribine-treated MoDCs proliferated more strongly, at lower DC/CD4⁺T-cell ratio (1:80), and secreted significantly higher levels of IL-17 and IFN-γ compared to the cultures with control MoDCs. The stimulatory effect of loxoribine on T helper (Th)1 polarization capability of MoDCs was further potentiated by ligation of CD40. In conclusion, our results show that loxoribine stimulated differentiation, maturation, allostimulatory as well as Th1 and Th17 polarization capability of human MoDCs and suggests that these effects might be associated with up-regulation of TLR7 expression, but not increased IFN-β production.     

Outer membrane protein A of Acinetobacter baumannii induces differentiation of CD4+ T cells toward a Th1 polarizing phenotype through the activation of dendritic cells

Acinetobacter baumannii is an increasing hospital-acquired pathogen that causes a various type of infections, but little is known about the protective immune response to this microorganism. Outer membrane protein A of A. baumannii (AbOmpA) is a major porin protein and plays an important role in pathogenesis. We analyzed interaction between AbOmpA and dendritic cells (DCs) to characterize the role of this protein in promoting innate and adaptive immune responses. AbOmpA functionally activates bone marrow-derived DCs by augmenting expression of the surface markers, CD40, CD54, B7 family (CD80 and CD86) and major histocompatibility complex class I and II. AbOmpA induces production of Th1-promoting interleukin-12 from DCs and augments the syngeneic and allogeneic immunostimulatory capacity of DCs. AbOmpA stimulates production of interferon-γ from T cells in mixed lymphocyte reactions, which suggesting Th1-polarizing capacity. CD4⁺ T cells stimulated by AbOmpA-stimulated DCs show a Th1-polarizing cytokine profile. The expression of surface markers on DCs is mediated by both mitogen-activated protein kinases and NF-κB pathways. Our findings suggest that AbOmpA induces maturation of DCs and drives Th1 polarization, which are important properties for determining the nature of immune response against A. baumannii.     

Cordlan polysaccharide isolated from mushroom Cordyceps militaris induces dendritic cell maturation through toll-like receptor 4 signalings

Defect of dendritic cell (DC) maturation in tumor microenvironments is an important immunological problem limiting the success of cancer immunotherapy. Here, we investigated the effects of polysaccharide (cordlan) isolated from Cordyceps militaris on DC maturation. Phenotypic maturation of DCs by cordlan was demonstrated by the elevated expressions of CD40, CD80, CD86, MHC-I, and MHC-II molecules, and functional maturation by increased expression of IL-12, IL-1β, TNF-α, and IFN-αβ, enhanced allogenic T cell stimulation, and decreased endocytosis. Of note was that cordlan induced maturation of tlr4^+/+ DCs from C3H/HeN mice, but not tlr4^−/− DCs from C3H/HeJ mice, suggesting the promising membrane receptor of cordlan. In addition, cordlan increased phosphorylation of ERK, p38, and JNK, and nuclear translocation of NF-κB p50/p65, which were main signaling molecules down-stream from TLR4. These results indicate that cordlan induces DC maturation through TLR4 signaling pathways.     

Analysis of the role of palmitoleic acid in acute anterior uveitis

PurposeTo study the role of palmitoleic acid (PA) in the pathogenesis of acute anterior uveitis (AAU).MethodsPA levels in feces from AAU patients were measured by gas chromatography coupled with a mass spectrometer (GC-MS) and compared with samples obtained from healthy individuals. Enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to assess the effect of PA on dendritic cells (DCs) and CD4⁺T cells obtained from mice, AAU patients and healthy individuals. C57BL/6 mice were fed with PA or vehicle and experimental autoimmune uveitis (EAU) was induced with a human retinal IRBP651-670 peptide. Disease severity of EAU was evaluated by clinical manifestation and histology. Differentiation of splenic Type 1 helper T cells (Th1) and Th17 cells was evaluated by FCM. Tandem mass tag (TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of DCs with PA.ResultsThe fecal concentration of PA was increased in AAU patients as compared with healthy individuals. In vitro, PA promoted apoptosis of DCs and inhibited the secretion of TNF-α from mouse bone-marrow-derived dendritic cells (BMDCs) as well as in DCs from AAU patients and healthy individuals. It only decreased DCs surface marker expression and IL-12p70 secretion in BMDCs and healthy individuals DCs but not in AAU patient DCs. PA-treated BMDCs inhibited Th cell differentiation from mouse naïve CD4⁺T cells and IL-17 and IFN-γ secretion in co-culture supernatants. PA also inhibited the differentiation of Th cells and secretion of IFN-γ and IL-17 in CD4⁺T cells from mice, AAU patients and healthy individuals. In vivo, PA-treated EAU mice showed milder clinical and histopathological intraocular manifestations as compared with the control group. PA feeding inhibited differentiation of splenic Th17 cells, whereas Th1 cells were not affected. Up to 30 upregulated and 77 downregulated proteins were identified when comparing PA-treated DCs with controls.ConclusionAn increased expression of fecal PA was observed in AAU patients. PA was shown to have immunoregulatory effects on DCs and CD4⁺T cells and attenuated disease severity in EAU mice.