Dendritic Cells Costimulatory Molecules Research Articles

Overexpression of FBP1 enhances dendritic cell activation and maturation by inhibiting glycolysis and promoting the secretion of IL33 in lung adenocarcinoma

Fructose 1,6-diphosphatase 1 (FBP1) is an enzyme involved in gluconeogenesis and glycolysis inhibition. Dendritic cells (DCs) are antigen-presenting cells, and antigens presented to T cells activate the immune response. FBP1 inhibits the development of several tumors, and high FBP1 expression inhibits the proliferation, migration, and invasion of lung cancer cells. However, the mechanism through which FBP1 mediates the tumor immune microenvironment is unclear. This study mainly analyzed the role of FBP1 in regulating the function of DCs through metabolic reprogramming and immune microenvironment using in vitro and in vivo experiments. The positive association of FBP1 with DCs was found by bioinformatic analysis. The in vitro experiments revealed that the extracellular acidification rate and lactate level were lower in the FBP1 overexpression cells than in the control cells and that the lower lactate level reduced the inhibition of DC function. In addition, high FBP1 expression promoted the secretion of IL33 by activating the cGAS/STING/NF-κB/IL33 pathway, which was identified and verified via high-throughput sequencing and in vitro experiments. FBP1 activated the cGAS/STING pathway by increasing the degree of DNA damage, as revealed by the level of γH2AX and comet assay. IL33 enhanced the expression of the DC costimulatory molecules CD86 and HLA-DR as well as that of the functional factor IL-1β. The results demonstrated that FBP1 promoted the activation and maturation of DCs by inhibiting glycolysis and promoting the secretion of IL33 as well as by further activating the function of CD8+T cells. Finally, the humanized immune system mouse models confirmed the above role of FBP1. Thus, FBP1 may serve as a new target to cure lung adenocarcinoma, and IL33 may improve the efficiency of immune therapy in lung adenocarcinoma.

Deciphering immunological mechanisms underlying the efficacy of nanoparticle-based vaccines against opioid use disorder (OUD)

Abstract The highly complex OUD and overdose epidemic poses a huge public health and economic burden, urging the need for safe and more effective therapeutic options. Vaccines offer a promising strategy for OUD treatment and prevention of overdose. Vaccinating against opioids generates drug-specific polyclonal antibodies (Ab) that selectively bind targeted opioids, blocking their distribution to the brain and thus blunting opioid induced rewards, pharmacological and side effects. Previous clinical trials of addiction vaccines showed proof of efficacy only in subjects with the highest Ab titers, highlighting the need to design more effective vaccines. The generation of a robust anti-drug polyclonal Abs response relies on CD4+ T cell-dependent B cell activation, hence dendritic cells (DC) are crucial for initiating CD4+ T cells priming and B cell responses. To improve vaccine formulation, we are exploiting a lipid-polymer hybrid nanoparticle (LPNP) platform and toll-like receptor (TLR) agonists to potentiate antigen recognition by antigen presenting cells (APCs) and subsequently the efficacy of anti-opioid conjugate vaccines. Using a flow cytometry-based in vitro activation assay, we demonstrated that LPNP-based vaccines are more effective in inducing macrophages activation marker (iNOS) and DCs co-stimulatory molecules and maturation markers (CD86, CD40, and MHC II) compared to conventional vaccines. Adding selected TLR agonists further improved nanovaccine efficacy in inducing the maturation and activation of APCs. These findings were confirmed with RT-qPCR quantifying mRNA expression of above-mentioned markers. These studies will identify key APCs subsets contributing to vaccine efficacy against OUD and other targets. Supported by grants from NIH (UG3/UH3 DA048775)

Dendritic cells and their associated pro-inflammatory cytokines augment to the inflammatory milieu in vitiligo skin

Vitiligo is a progressive, autoimmune, hypomelanotic acquired disorder of skin which is characterized by depigmentation. The initial immunological events of this diseases are still at enigma that includes breach of immune tolerance, and defect in antigen presentation. Hence, we aimed to explore role of Dendritic cells (DCs) and its associated cytokines in the pathogenesis of generalized vitiligo (GV) patients. For this case-control study, 20 active patients and controls were enrolled. Phenotypic characterization of myeloid and plasmacytoid DCs (mDCs, pDCs) were done by flow-cytometry. Primary culture of DCs was done by monocyte differentiation supplemented with rIL-4 and rGM-CSF. Functional analysis DCs related cytokines and co-stimulatory molecules (CD80, CD40) was done by ELISA and qPCR respectively. Tissue localization of DCs was evaluated by immunohistochemistry. The frequency of mDCs (0.3715% v/s 0.188%) and pDCs (0.2331% v/s 0.1156%) were elevated in patients as compared to controls. Circulatory level of IL-12, TNF-α were significantly higher whereas IFN-α was decreased in patients than controls. Similar results were obtained in the culture supernatants of patients. Relative mRNA expression profiling of co-stimulatory molecules (CD80, CD40) were found to be up regulated in patient's skin. Tissue localization of Langerhans cells (Langerin, CD1a+) were found to be significantly higher in patients. Elevated frequency of mDCs and pDCs along with elevated levels of IL-12, TNF-α and CD80, CD40 may contribute in defective antigen presentation of DCs. Altered pro-inflammatory and anti-inflammatory cytokines along with tissue localization of Langerhans cells might be involved in the pathogenesis of GV. These DCs associated cytokines can be explored as a therapeutic target in future.

Ablative Radiotherapy Synergizes With Protein Vaccination to Upregulate Systemic Dendritic Cell Function Within Non-Draining Lymph Nodes

Dendritic cells (DCs) play a critical role in the response of T cells to soluble antigen. Dendritic cells instruct whether initial T cell priming interactions result in anti-tumor immunity or tolerance. Type I conventional dendritic cells (cDC1) are required in order to prime functional anti-tumor CD8 T cell responses through cross-presentation of extracellular tumor associated antigens. In tumor bearing mouse models, radiotherapy (RT) has been shown to stimulate cDC1 dependent cytotoxic T cell responses. Here we utilize a tumor free mouse model system to interrogate the effects of RT on DCs within draining and distant lymph nodes. Our hypothesis is that ablative radiotherapy will synergize with immunomodulation (Poly I:C) to systemically increase the function of DCs within both draining and abscopal sites.Utilizing a vaccine model to evaluate the effects of ablative radiotherapy on DC percentage, phenotype, and function, C57BL/6 mice were subcutaneously vaccinated in the flank with soluble ovalbumin (OVA) antigen with or without Poly I:C adjuvant and followed by a single 20 Gy fraction of RT at the vaccine site. Antigen uptake was evaluated using AlexFluor-488 conjugated OVA and antigen processing by DCs was assessed using DQ-OVA, a self-quenching protein conjugate which only exhibits fluorescence upon proteolytic degradation. Following treatment, draining and non-draining lymph nodes with respect to RT were evaluated separately by flow cytometry to assess dendritic cell maturation, antigen load, and antigen processing.We found that cDC1 within lymph nodes draining a vaccination site receiving OVA, Poly I:C and RT had increased antigen processing and increased expression of DC licensing and costimulatory molecules compared to either vaccination or RT alone. We also discovered that cDC1 in non-draining (abscopal) nodes did not receive trafficked antigen from the irradiated or Poly I:C vaccine site; however, they did demonstrate increased uptake and processing of local antigen when Poly I:C and RT were co-administered at distal sites. Therefore, the combination of RT and vaccination resulted in systemic increases in DC function distant from the RT site.Ablative RT delivered with soluble antigen and Poly I:C adjuvant results in increased systemic function of cDC1 compared to vaccination or RT alone. This experimental system reveals that radiotherapy synergizes with immunomodulation to increase systemic antigen uptake and processing by the DC population required for priming of functional anti-tumor immunity. This work has implications regarding how RT to a single lesion may facilitate priming or reactivation of distal anti-tumor immune responses in metastatic cancer. Follow up studies are underway to elucidate the mechanism of this abscopal effect on DC function and evaluate whether local radiotherapy results in increased tumor associated antigen uptake and presentation at distant sites.

Inhibition of Dectin-1 on Dendritic Cells Prevents Maturation and Prolongs Murine Islet Allograft Survival.

IntroductionThe ability of dendritic cells (DCs) to initiate an immune response or induce immune tolerance depends on their maturation status. Dendritic-cell-associated C-type lectin 1 (Dectin-1) plays a key role in the differentiation, activation, and maturation of DCs. Therefore, we hypothesized that inhibition of Dectin-1 could prevent DC maturation and induce immune tolerance of transplanted organs.MethodsDCs were transduced with a recombinant lentiviral vector to inhibit Dectin-1 and then were injected into a murine recipient before islet transplantation. C57BL/6 mice (H-2b) were treated with lentiviral vector-Dectin-1-RNAi-DC (DC-Dectin-1-RNAi group), lentiviral vector-GFP DCs (DC-GFP group), and PBS (control group). Pancreatic islet transplantation was performed and graft survival was recorded. The proportions of regulatory T cells, Th1 cells, and Th17 cells in the spleen and draining lymph nodes, and serum levels of interleukin (IL)-10, IL-17, and interferon (INF)-γ were measured.ResultsThe inhibition of Dectin-1 resulted in low expression of MHC-II and costimulatory molecules in DCs. Murine recipients treated with DC-Dectin-1-RNAi had longer islet allograft survival time, a reduction in the levels of Th1 and Th17 cells and secreted cytokines, and an increase of Treg cells.ConclusionThe inhibition of Dectin-1 by recombinant lentiviral vector Dectin-1-RNAi inhibits the maturation and activation of DCs, affects the differentiation of T cell subsets, and prolongs allograft survival.

Proof of Concept Study: Mesoporous Silica Nanoparticles, From Synthesis to Active Specific Immunotherapy

Nanomaterials are increasingly valued tools in drug delivery research as they offer enhanced stability, controlled release and more effective drug encapsulation. Though yet to be introduced in clinical trial, mesoporous silica nanoparticles are promising delivery systems, due to their high chemical and mechanical stability while remaining biodegradable. This work provides proof of concept for particle based vaccines as cost-effective alternatives for dendritic cell immunotherapy. Synthesis and surface chemistry of the nanoparticles are optimized for protein conjugation and nanoparticles are characterized for their physicochemical properties and biodegradation. Ovalbumin is used as a model protein to load nanoparticles to produce a nanovaccine. The vaccine is tested in vitro on dendritic cultures to verify particle and vaccine uptake, toxicity, maturation effects and explicitly ovalbumin cross-presentation on MHC class I molecules. The optimized synthesis protocol renders reproducible mesoporous silica nanoparticles, resistant against agglomeration, within the required size range and have carboxylic surface functionalization necessary for protein conjugation. They are biodegradable over a time span of 1 week. This period is adjustable by changing synthesis parameters. UV sterilization of the particles does not induce quality loss, nor does it have toxic effects on cells. Treatment with mesoporous silica nanoparticles increases expression of MHC and costimulatory molecules of dendritic cells, indicating an adjuvant effect of nanoparticles on the adaptive immune system. Nanovaccine uptake and cross-presentation of ovalbumin are observed and the latter is increased when delivered by nanoparticles as compared to control conditions. This confirms the large potential of mesoporous silica nanoparticle based vaccines to replace dendritic-based active specific immunotherapy, offering a more standardized production process and higher efficacy.

Laser-assisted skin delivery of immunocontraceptive rabies nanoparticulate vaccine in poloxamer gel

A painless skin delivery of vaccine for disease prevention is of great advantage in improving compliance in patients. To test this idea as a proof of concept, we utilized a pDNA vaccine construct, pDNAg333-2GnRH that has a dual function of controlling rabies and inducing immunocontraception in animals. The pDNA was administered to mice in a nanoparticulate form delivered through the skin using the P.L.E.A.S.E.® (Precise Laser Epidermal System) microporation laser device. Laser application was well tolerated, and mild skin reaction was healed completely in 8 days. We demonstrated that adjuvanted nanoparticulate pDNA vaccine significantly upregulated the expression of co-stimulatory molecules in dendritic cells. After topical administration of the adjuvanted nano-vaccine in mice, the high avidity serum for GnRH antibodies were induced and maintained up to 9 weeks. The induced immune response was of a mixed Th1/Th2 profile as measured by IgG subclasses (IgG2a and IgG1) and cytokine levels (IFN-γ and IL-4). Using flow cytometry, we revealed an increase of CD8+ T-cells and CD45R B cells upon the administration of the adjuvanted vaccine. Our previous study used the same pDNA nanoparticulate vaccine through an IM route, and a comparable immune response was induced using P.L.E.A.S.E. However, the vaccine dose in the current study was four-fold less than what was applied through the IM route.We concluded that laser-assisted skin vaccination has a potential of becoming a safe and reliable vaccination tool for rabies vaccination in animals or even in humans for pre- or post-exposure prophylaxis.

CD40 up-regulation on dendritic cells correlates with Th17/Treg imbalance in chronic periodontitis in young population

We aimed to discover the influence of age on the development of chronic periodontitis and illustrate the molecular mechanism in this process. Blood samples were collected from 63 chronic periodontitis patients and 30 healthy controls. Th17 cell/Foxp3+ regulatory T cell (Treg) ratio and expression of costimulatory molecules in dendritic cells (DCs) were analyzed by flow cytometry. The serum levels of soluble CD40 ligand (CD40L) and IL-17 were examined by ELISA. In young chronic periodontitis patients, the Th17/Treg ratio was significantly higher than that in old patients. CD40 on DCs and serum levels of CD40L and IL-17 were all higher in young chronic periodontitis patients. Mature DCs with high CD40 expression level elevated the Th17/Treg ratio in vitro. During the pathogenesis of chronic periodontitis, young patients had higher Th17/Treg ratio than old patients and this phenomenon was in line with the differential expression levels of CD40 in DCs.

Abstract LB-192: The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression of CD103+ dendritic cells

Abstract Adenosine signaling is a normal physiologic process preventing autoimmunity, that is co-opted by tumors as an immune escape mechanism. Dendritic cells (DC), generated from human monocytes or mouse bone marrow, are susceptible to the immunosuppressive effects of extracellular adenosine. In vitro studies using a stable analog of adenosine, 5’-N-ethylcarboxamidoadenosine (NECA), demonstrate DC dysfunction in differentiation and of priming naïve T cells. This negatively effects the critical role of DCs to drive antigen-specific tumor cytotoxic T cell responses under conditions of high extracellular adenosine. AZD4635 (HTL-1071) is a selective oral A2AR antagonist, currently in clinical trials as a single agent and in combination with durvalumab (anti-PD-L1 Ab) in patients with solid malignancies. We have reported previously that AZD4635 treatment led to reduction in tumor growth as monotherapy and in combination with anti-PD-L1 Ab in several syngeneic mouse tumor models. Antitumor activity by AZD4635 was supported by the observation that AZD4635 treated tumors had increased expression of genes associated with immune activation and increased expression of co-stimulatory molecules on antigen presenting cells (APCs). We now extend our previous findings by demonstrating improved antigen presentation in antigen-specific mouse (OVA) and human (Melan-A) systems and show reversed dysfunction in mouse CD103+ crosspresenting/crosspriming DC. In this report, we tested the ability of AZD4635 to prevent adenosine-mediated immunosuppression of antigen specific responses in both mouse and human DCs. We demonstrate that AZD4635 could significantly prevent NECA-mediated immuno-suppression of critical aspects of CD103+ DC phenotype and function in vitro. These included CD103+ DC differentiation and costimulatory molecule expression in FLT-3L/GM-CSF differentiated mouse bone marrow cultures, reduced pinocytosis of ovalbumin and subsequently reduced antigen processing and presentation, leading to weaker OT-I T cell functional responses in vitro. NECA also affected the ability of CD103+ DC to crosspresent antigens from dying MC38-Ova tumor cells and crossprime OT-I T cells in vitro. In vivo, immunophenotyping showed that mice bearing MC38-OVA tumors treated with AZD4635 and anti-PD-L1 had increased antigen-specific T cells and CD103+ DC in tumors. CD103+ DC from TdLN of AZD4635 treated mice elicited proliferation of OT-I T cells without exogenously added antigen. In co-cultures of naïve human CD8+ T cells and DCs containing an HLA-A2-restricted Melan-A peptide, NECA abrogated the ability of monocyte-derived DC to prime antigen specific CD8+ T cells. Adenosine antagonism by AZD4635 improved antigen presentation and costimulation by DCs, leading to better priming and expansion of antigen specific T cells. In summary, we report that AZD4635’s MOA includes restoration of DC function in the elicitation of antigen-specific T cell responses. Citation Format: Christine M. Barbon, Alexandra Borodovsky, Yanjun Wang, Laura Prickett, Kris Sachsenmeier, Alwin Schuller, Wenlin Shao, Carl Barrett, Stephen Fawell, Deanna A. Mele. The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression of CD103+ dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-192.

Coronary artery bypass grafting surgery is associated with immunoparalysis of monocytes and dendritic cells

Abstract Background Exposure of patient blood to the bypass circuitry and re-perfusion of the heart at the end of coronary artery bypass grafting (CABG) is associated with activation of leucocytes, the complement cascade and cytokine secretion. Dendritic cells (DC) and monocytes are immunoregulatory cells that may be impacted by CABG contributing to an altered immune status. This study investigated changes in the DC and monocyte immune profile in CABG patients at 5 time-points: admission, peri-operative, ICU, day 3 (D3) and day 5 (D5). Methods At each time point, freshly collected whole blood from 50 CABG patients was used in an ex-vivo whole blood culture model. Lipopolysaccharide (LPS) was added in parallel to model an infectious complication. Expression of DC and monocyte co-stimulatory and adhesion molecules and production of intracellular inflammatory mediators was measured via flow cytometry. Results CABG resulted in significant perturbation of monocyte and DC responses. DC and monocytes were immunoparalysed as evidenced by failure to respond to LPS stimulation. Modulation of DC and monocyte costimulatory and adhesion molecule expression and inflammatory mediator production was associated with prolonged length of stay in intensive care and post-operative atrial fibrillation. While DC and monocyte expression of co-stimulatory and adhesion molecules recovered towards baseline by D5 post-surgery, their capacity to produce inflammatory mediators did not. Conclusions DC and monocyte responses were significantly impaired following CABG. Use of an ex-vivo model to assess immune competency of cardiac surgery patients may provide a tool for the early prediction of adverse outcomes.

Antigen-loaded dendritic cells triggers a specific cytotoxic Tlymphocytes immune response against hepatocellular carcinoma: in vitro study.

Hepatocellular carcinoma (HCC) is one of the common malignancies, characterized by poor response to conventional therapeutic options. Immunotherapy with dendritic cells (DCs)-vaccines is one of the most successful strategies used for the treatment of HCC. However, the methods applied in the preparation of antigen-loaded DCs are important factors for optimization of DCs vaccines. The present study was conducted to investigate the effect of HCC-whole tumor cell lysate prepared using rapid repetitive freeze-thaw cycles on the immunogenicity of DCs and evaluate the ability of whole tumor cell lysate-pulsed DCs vaccine to induce a specific cytotoxic Tlymphocytes (CTLs) response against HepG2 cell line. Immature DCs generated from peripheral blood monocytes were randomized into two groups: control DCs and whole tumor cell lysate-pulsed DCs. Phenotypic analysis of the DCs' cell maturation marker CD83 and co-stimulatory molecule CD86 was performed. HCC-specific cytotoxic activity of CD8+ CTLs was measured in vitro. Loading of DCs with necrotic whole cell lysate resulted in non-significant changes in DCs' expression of CD83, but a significant increase in expression of CD86. In addition, CD8+ CTLs stimulated with whole tumor cell lysate-pulsed DCs showed a high cytotoxic activity that specifically attack HepG2 cells. Our findings indicated that pulsation of DCs with whole tumor cell lysate prepared by repetitive freeze-thaw cycles could efficiently enhance the ability of DCs to induce proliferation and clonal expansion of CD8+ CTLs. Data herein, also indicated that whole tumor cell lysate-pulsed DCs triggers a specific CD8+ CTLs against HCC tumor cells.

Trichostatin a Protects Dendritic Cells Against Oxygen-Glucose Deprivation via the SRSF3/PKM2/Glycolytic Pathway.

Dendritic cells (DCs) are important to the immune system and are frequently recruited to hypoxic regions, especially during acute myocardial infarction (AMI). Emerging data indicate that histone deacetylase (HDAC) inhibitors possess immunomodulatory functions. We previously showed in a rat model of AMI that the HDAC inhibitor TSA improved tissue repair, and this was accompanied by increased DC infiltration in the infarct region, suggesting an important role of TSA in modulating DC functions. To study the potential modulatory effect of TSA on DCs, we exploited an in vitro model of hypoxia and glucose deprivation. Culturing of DCs in the presence of 200 nM TSA improved DC survival under hypoxia and glucose deprivation. However, on a phenotypic level, TSA induced the expression of the DC co-stimulatory molecules CD80 and CD86, decreased FITC-dextran uptake, and facilitated DC migration. Moreover, TSA altered cytokine secretion by reducing the pro-inflammatory cytokines IL-1β, IL-10, IL-12, and TGF-β. Furthermore, TSA treatment enhanced HIF-1α-dependent glycolytic gene expression and increased pyruvate kinase M2 by upregulating SRSF3. These results suggest that by TSA alters important DC functions under hypoxia and glucose deprivation, and that TSA is critical for DC function by modulating SRSF3-PKM2-dependent glycolytic pathways.

Programmed Cell Death Protein 1-PDL1 Interaction Prevents Heart Damage in Chronic Trypanosoma cruzi Infection.

Chagas disease is a neglected parasitic infection that affects around six to seven million people, mainly in Latin America. About 30–35% of infected people present chronic Chagas cardiomyopathy (CCC), which eventually leads to death. This condition is characterized by local parasite persistence and leukocyte infiltration. In a murine model of CCC, we observed that among infiltrating leukocytes, CD4+ and CD8+ T cells were in higher frequency in the heart of chronically infected mice, although elevated expression of the regulatory molecules programmed cell death protein 1 (PD1) and PDL1 suggested these cells could be inhibited. To investigate if PD1–PDL1 interaction in the heart of chronically infected mice negatively impacts on the local immune response, facilitating parasite persistence, and progression to CCC, we attempted to recover the local immune response by treating chronically infected mice with anti-PD1 and anti-PDL1-blocking antibodies together with irradiated Trypanosoma cruzi, which provides immune response boosting. Irradiated parasites promote expression of costimulatory molecules in dendritic cells and provide specific parasite antigen, which should aid T cell reactivation upon checkpoint blockade. Following treatment, there was an increased frequency of heart-infiltrating CD4+ and CD8+ T cells with an effector memory phenotype, an increased histopathology score and decreased heart rate, supporting our previous hypothesis of local immunosuppression induced by this pathway during CCC. In addition, blood parasitemia was reduced, which was associated with increased T. cruzi-specific immunoglobulin G 1 antibodies. However, no difference was observed in cytokine production or T. cruzi burden in the hearts of treated mice. Taken together, our results suggest PD1–PDL1 interaction protects the heart from excessive immune response.

The immunosuppressive effects of CD4+ CD25+ regulatory T cells on dendritic cells in patients with chronic hepatitis B.

The characteristics and functions of CD4+ CD25+ regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4+ CD25+ Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4+ CD25+ Tregs on the maturation and function of monocyte-derived DCs were examined. The results showed that CD4+ CD25+ render the DCs inefficient as antigen-presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL-10 and TGF-β. There were an increased IL-10 and TGF-β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4+ T cells, CD4+ CD25+ may modulate the immune response through DCs in CHB patients.

Digital gene expression profiling of dendritic cells treated with Seabuckthorn favones

Objective To study the changes in morphology, phenotypes and gene expression profiles of dendritic cells (DCs) following treatment with Seabuckthorn flavones (SF). Methods DCs were treated with 200 μg/ml of SF and then cultured for 7 days. Changes in the morphology of DCs were observed under light microscope. Flow cytometry was used to detect DC surface molecules. Total RNA was extracted to construct the library for digital gene expression profiling (DGE). Differentially expressed genes were screened out and further analyzed by gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Results Compared with control group, SF treatment significantly enhanced the expression of HLA-DR, CD80, CD83 and CD86 on DCs. A total of 355 differentially expressed genes were screened out by DGE, including 176 up-regulated genes and 179 down-regulated genes. GO enrichment was mainly involved in the regulation and development of the immune system and other biological processes. KEGG pathway analysis showed that the significantly enriched pathways were closely related to inflammation, the immune system, cancer and other diseases. Conclusion SF can promote the expression of DC co-stimulatory molecules and pro-mature molecules, and regulate the expression of immunity-related genes such as CD11a, SLAMF6, LMCD1, TSC22D3 and IKZF3. Key words: Seabuckthorn flavone; Dendritic cell; Digital gene expression profile (DGE); Immunoregulation

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses.The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4+ and CD8+ T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4+ and CD8+ lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized.DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30–40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4+ and CD8+ T cells, in vivo.Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects.

Downregulation of L-arginine metabolism in dendritic cells induces tolerance to exogenous antigen.

Dendritic cells (DC) are potential tools for therapeutic applications and several strategies to generate tolerogenic DCs are under investigation. When activated by cytokines and microbial products, DCs express mediators that modulate immune responses. In this regard, the metabolites generated by the activities of inducible nitric oxide synthase (iNOS) and arginase in DCs seem to play important roles. Here, we evaluated the effects of adoptive transfer of DCs generated in vitro from bone marrow precursors (BMDC) modulated with L-NAME (Nω-nitro-L-arginine methyl ester) and NOHA (NG-Hydroxy-L-arginine), inhibitors of iNOS and arginase, respectively, upon the immune response of the wild type (BALB/c) and OVA-TCR transgenic (DO11.10) mice. The modulation with L-NAME increased CD86 expression in BMDC, whereas treatment with NOHA increased both CD80 and CD86 expression. Adoptive transfer of either L-NAME- or NOHA-modulated BMDCs to BALB/c mice reduced the plasma levels of ovalbumin-specific antibody as well as proliferation and cytokine secretion in cultures of spleen cells in comparison adoptive transfer of non-modulated DCs. Conversely, transfer of both modulated and non-modulated BMDCs had no effect on immune response of DO11.10 mice. Together, these results show that the treatment with iNOS and Arg inhibitors leads to increased expression of co-stimulatory molecules in DCs, and provides evidences that L-arginine metabolism may be an important therapeutic target for modulating immune responses in inflammatory disorders.

Effects of RNA Interference on CD70 in Dendritic Cells of Patients with Immune Thrombocytopenia

Objective: This study aims to assess the biological properties and functions of CD70 costimulatory molecule on dendritic cells (DCs) from patients with chronic immune thrombocytopenia (ITP).Methods: DCs were generated from monocytes isolated from peripheral blood, the immunophenotype markers were detected by flow cytometry. Chemically synthesized siRNA was then transferred into the cells by Lipofectamine 2000 to choose the most effective siRNA to block CD70 expression in three candidates. The mRNA expression and protein synthesis were analyzed by real-time RT-PCR and flow cytometry. The CD4+ T cells were co-cultured with DCs to assess the suppression capacity of DCs in the proliferation of CD4+ T cells and the production of IL-10 and IFN-γ by CD4+ T cells. The CD4+ T cells were co-cultured with DCs to assess the ability of DCs in the induction of Regulatory T cells (Tregs).Results: Dendritic cells were transfected with FITC-labelled scrambled siRNA for 4-6h. The transfection efficiency was average 60%. After transfection (48 h), the results of real-time RT-PCR analysis showed strong and specific downregulation of CD70 mRNA levels in transfected cells (p=0.046, siRNA1\\2\\3 Inhibition rates (74.5±6.1) %, (78.1±8.0) %, (90.1±2.5) % respectively, but not in negative control or in the absence of siRNA(p=0.157). The CD70-3 siRNA was extraordinarily effective in repressing the expression of CD70. The expression of CD70 on the DCs with siRNA3 was also significantly lower(p=0.043), but showed no difference in negative control (p=0.157). DCs from transfected group could inhibit the proliferation of PHA-activated CD4+ T cells than the negative control in ITP patients(p=0.008), but showed no difference in group of normal control (p=0.08). In chronic ITP patients and normal controls, the DCs from transfected group could inhibit the expression of IFN-γ compared with the group of negative control (p=0.018, p=0.043) and increase the expression of IL-10 (p=0.012, p=0.043). In chronic ITP patients and normal controls, the DCs from transfected group were defective in inducing the CD4+ T cell differentiating to CD4+CD25+CD127low Tregs compared to the negative control (p=0.012, p=0.043).Conclusions: In our study, siRNA is capable of inducing RNAi in DCs, it can knock down the CD70 gene expression specifically and effectively. The DCs from transfected group could inhibit the CD4+ T cells and induce the CD4+ T cells differentiating to Tregs. This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for autoimmune disease. DisclosuresNo relevant conflicts of interest to declare.

Curcumin Suppressed Activation of Dendritic Cells via JAK/STAT/SOCS Signal in Mice with Experimental Colitis.

Dendritic cells (DCs) play a pivotal role as initiators in the pathogenesis of inflammatory bowel disease and are regulated by the JAK/STAT/SOCS signaling pathway. As a potent anti-inflammatory compound, curcumin represents a viable treatment alternative or adjunctive therapy in the management of chronic inflammatory bowel disease (IBD). The mechanism of curcumin treated IBD on DCs is not completely understood. In the present study, we explored the mechanism of curcumin treated experimental colitis by observing activation of DCs via JAK/STAT/SOCS signaling pathway in colitis mice. Experimental colitis was induced by 2, 4, 6-trinitrobenzene sulfonic acid. After 7 days treatment with curcumin, its therapeutic effect was verified by decreased colonic weight, histological scores, and remitting pathological injury. Meanwhile, the levels of major histocompatibility complex class II and DC costimulatory molecules (CD83, CD28, B7-DC, CD40, CD40 L, and TLR2) were inhibited and followed the up-regulated levels of IL-4, IL-10, and IFN-γ, and down-regulated GM-CSF, IL-12p70, IL-15, IL-23, and TGF-β1. A key finding was that the phosphorylation of the three members (JAK2, STAT3, and STAT6) of the JAK/STAT/SOCS signaling pathway was inhibited, and the three downstream proteins (SOCS1, SOCS3, and PIAS3) from this pathway were highly expressed. In conclusion, curcumin suppressed the activation of DCs by modulating the JAK/STAT/SOCS signaling pathway to restore immunologic balance to effectively treat experimental colitis.

Modulation of Proinflammatory Cytokines in Monocyte-Derived Dendritic Cells by Porcine Reproductive and Respiratory Syndrome Virus Through Interaction with the Porcine Intercellular-Adhesion-Molecule-3-Grabbing Nonintegrin.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important global swine pathogen. PRRSV infects porcine dendritic cells (DCs), but the effects of the interactions with DCs are largely unknown. Current research focuses on the production and regulation of interferons and selected inflammatory cytokines in DCs, which may play key roles in immune modulation. In addition, PRRSV also downregulates swine leukocyte antigen class I (SLA-I), SLA-II, and CD80/86 costimulatory molecules in DCs. In this study, we aim to evaluate the PRRSV immunomodulatory effects on monocyte-derived DCs (MoDCs) through interactions with porcine DC-SIGN (pDC-SIGN) receptor. We demonstrated that blocking the PRRSV and pDC-SIGN interactions in MoDCs with recombinant hICAM-3 did not affect the regulatory effects of PRRSV on SLA-I, SLA-II, or CD80/86 molecules. The hICAM-3 did not affect the morphological changes on MoDCs associated with their activation and maturation after PRRSV infection, and did not impair the virus infectivity in these cells either. The mRNA levels of tumor necrosis factor alpha (TNF-α), IL-12p35, IL-1β, and IL-6 were upregulated after hICAM-3 treatment or PRRSV infection, but in the presence of the blockage of pDC-SIGN in MoDCs with hICAM-3, PRRSV did not modulate the expression of these genes. However, in the presence of an anti-pDC-SIGN monoclonal antibody (mAb), we showed that PRRSV infection significantly reduced the mRNA expression levels of TNF-α and IL-1α, but enhanced the expression of IL-12p35 in MoDCs. Both hICAM-3-Fc and pDC-SIGN mAb treatments did not modulate proinflammatory cytokine protein levels in the culture supernatants of PRRSV-infected MoDCs. The results indicate that blocking the PRRSV-pDC-SIGN interactions by recombinant hICAM-3-Fc did not significantly affect virus infectivity, DC maturation, and proinflammatory cytokine gene expression in infected MoDCs. However, blocking the PRRSV-pDC-SIGN interactions on MoDCs with an anti-pDC-SIGN mAb revealed differential regulatory effects on specific proinflammatory gene expressions in those cells.