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The purpose of this study was to develop a monoclonal antibody (mAb) that can identify porcine dendritic cells (DCs) that have differentiated from bone marrow progenitor cells. Hybridoma technology was used to obtain mAbs, and bone marrow-derived DCs (BMDCs) were employed as immunogens for producing antibodies. The generated PD9-9 mAbs exhibited considerable reactivity towards porcine BMDCs with applications in flow cytometry and immunostaining. The antibody was composed of heavy immunoglobulin gamma-1 chains and light kappa chains. The PD9-9 mAb recognized fully differentiated porcine BMDCs and cells undergoing DC differentiation. In contrast, bone marrow cells and macrophages were not recognized by PD9-9. In addition, the PD9-9 mAb promoted porcine DC proliferation. Consequently, the PD9-9 mAb may be a biomarker for porcine DCs and will be advantageous for investigating porcine DC biology. [ABSTRACT FROM AUTHOR]

To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow‐derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC‐mediated CTL priming, and further investigate the role of PI3K‐Akt engaged in K33O‐increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC‐based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R‐mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K‐Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC‐based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme‐linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K‐Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K‐Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC‐mediated CTL priming in bone marrow‐derived dendritic cells via PI3K‐Akt signalling. [ABSTRACT FROM AUTHOR]

Introduction: Inactivated parapoxvirus ovis (iPPVO) exerts strong immunomodulatory effects on innate immune cells, making it an attractive therapeutic candidate. However, little is known about the signaling pathways that are involved in iPPVO-induced immune responses. Methods: In this study, we systematically analyzed how different types of dendritic cells (DCs) react to iPPVO (Zylexis, strain D1701) in both BALB/c and C57BL/6 mice by flow cytometry and ELISAs, and investigated which signaling pathway is related to DC activation by Western blotting and protein profiling. Results: We demonstrated that bone marrow-derived conventional DCs (BM-cDCs) and bone marrow-derived plasmacytoid DCs (BM-pDCs) matured and secreted type I interferons in response to Zylexis stimulation in both mouse strains. Similarly, Zylexis promoted the secretion of IL-12/23p40 and TNF by pDCs. However, IL-12/23p40 and TNF secretion by cDCs were induced in BALB/c mice but not in C57BL/6 mice. Analyzing the underlying signaling pathways revealed that iPPVO-induced maturation of cDCs was Toll-like receptor 9 (TLR9) independent, while the maturation of pDCs partially depended on the TLR9 pathway. Moreover, the production of proinflammatory cytokines by cDCs and the secretion of IFN-α/β by pDCs partially depended on the TLR9 pathway in both mouse strains. Therefore, other signaling pathways seem to participate in the response of DCs to iPPVO, supported by protein profiling. Conclusion: Our data provide useful insights into the diversity of iPPVO sensors and their varying effects across different strains and species. [ABSTRACT FROM AUTHOR]

Background: Chemical‐induced allergies at workplace represent a significant occupational health issue. These substances must be properly identified as sensitizers. In previous studies, an original model using mouse bone marrow‐derived dendritic cells (BMDC) was developed for this purpose. Objectives: The aim of this study was to evaluate the predictive capacity of the BMDC model with a large panel of sensitizers (including pre‐ and pro‐haptens) and non‐sensitizers. Methods: The readout from the BMDC model is based on expression levels of six phenotypic markers measured by flow cytometry. Results: The results indicate that 29 of the 37 non‐sensitizers, and 81 of the 86 sensitizers were correctly classified compared to the Local Lymph Node Assay (LLNA). Statistical analysis revealed the BMDC model to have a sensitivity of 94%, a specificity of 78%, and an accuracy of 89%. The EC2 (Effective Concentration) values calculated with this model allow sensitizers to be categorized into four classes: extreme, strong, moderate and weak. Conclusions: These excellent predictive performances show that the BMDC model discriminates between sensitizers and non‐sensitizers with outstanding precision equal to or better than existing validated alternative models. Moreover, this model allows to predict sensitization potency of chemicals. The BMDC test could therefore be proposed as an additional tool to assess the sensitizing potential and potency of chemicals. [ABSTRACT FROM AUTHOR]

Background: Bisphenol (BP‐)A is a chemical used in Europe to produce polycarbonate plastics and epoxy resin or as colour developer in thermal paper. Due to its toxicity, BPA presence was restricted by European regulations. Therefore, substitute chemicals are replacing BPA. Objective: To assess the allergenic sensitizing potential of 27 substitutes to BPA used in the industry. Methods: The expression of two costimulatory molecules and six cytokines were analysed by flow cytometry in mouse bone marrow‐derived dendritic cells (BMDCs) exposed to the chemicals. Results: All substances except one induced overexpression of at least one receptor and were thus identified as having allergenic sensitizing potential. Based on the BMDC model, they were classified as extreme (1 out of 27), strong (20 out of 27) and moderate (5 out of 27) sensitizers. BPA was classified as a moderate sensitizer and BPF was the only substitute classified as a non‐sensitizer. The more potent substitutes induced more than 2‐fold secretion of CCL3, CCL4 and/or CCL5 by dendritic cells. Conclusion: Most of the BPA substitutes tested in this study have an allergenic sensitizing potential; 24 of them being more potent than BPA itself. Only BPE, BPF and 2,4‐BPS appeared to be weaker sensitizers than BPA. [ABSTRACT FROM AUTHOR]

The purpose of this study was to develop a monoclonal antibody (mAb) that can identify porcine dendritic cells (DCs) that have differentiated from bone marrow progenitor cells. Hybridoma technology was used to obtain mAbs, and bone marrow-derived DCs (BMDCs) were employed as immunogens for producing antibodies. The generated PD9-9 mAbs exhibited considerable reactivity towards porcine BMDCs with applications in flow cytometry and immunostaining. The antibody was composed of heavy immunoglobulin gamma-1 chains and light kappa chains. The PD9-9 mAb recognized fully differentiated porcine BMDCs and cells undergoing DC differentiation. In contrast, bone marrow cells and macrophages were not recognized by PD9-9. In addition, the PD9-9 mAb promoted porcine DC proliferation. Consequently, the PD9-9 mAb may be a biomarker for porcine DCs and will be advantageous for investigating porcine DC biology.

Introduction: Inactivated parapoxvirus ovis (iPPVO) exerts strong immunomodulatory effects on innate immune cells, making it an attractive therapeutic candidate. However, little is known about the signaling pathways that are involved in iPPVO-induced immune responses. Methods: In this study, we systematically analyzed how different types of dendritic cells (DCs) react to iPPVO (Zylexis, strain D1701) in both BALB/c and C57BL/6 mice by flow cytometry and ELISAs, and investigated which signaling pathway is related to DC activation by Western blotting and protein profiling. Results: We demonstrated that bone marrow-derived conventional DCs (BM-cDCs) and bone marrow-derived plasmacytoid DCs (BM-pDCs) matured and secreted type I interferons in response to Zylexis stimulation in both mouse strains. Similarly, Zylexis promoted the secretion of IL-12/23p40 and TNF by pDCs. However, IL-12/23p40 and TNF secretion by cDCs were induced in BALB/c mice but not in C57BL/6 mice. Analyzing the underlying signaling pathways revealed that iPPVO-induced maturation of cDCs was Toll-like receptor 9 (TLR9) independent, while the maturation of pDCs partially depended on the TLR9 pathway. Moreover, the production of proinflammatory cytokines by cDCs and the secretion of IFN-α/β by pDCs partially depended on the TLR9 pathway in both mouse strains. Therefore, other signaling pathways seem to participate in the response of DCs to iPPVO, supported by protein profiling. Conclusion: Our data provide useful insights into the diversity of iPPVO sensors and their varying effects across different strains and species.

ABSTRACTFormyl peptide receptor-1 (FPR1) is a pattern recognition receptor that is mostly expressed by myeloid cells. In patients with colorectal cancer (CRC), a loss-of-function polymorphism (rs867228) in the gene coding for FPR1 has been associated with reduced responses to chemotherapy or chemoradiotherapy. Moreover, rs867228 is associated with accelerated esophageal and colorectal carcinogenesis. Here, we show that dendritic cells from Fpr1−/− mice exhibit reduced migration in response to chemotherapy-treated CRC cells. Moreover, Fpr1−/− mice are particularly susceptible to chronic ulcerative colitis and colorectal oncogenesis induced by the mutagen azoxymethane followed by oral dextran sodium sulfate, a detergent that induces colitis. These experiments were performed after initial co-housing of Fpr1−/− mice and wild-type controls, precluding major Fpr1-driven differences in the microbiota. Pharmacological inhibition of Fpr1 by cyclosporin H also tended to increase intestinal oncogenesis in mice bearing the ApcMin mutation, and this effect was reversed by the anti-inflammatory drug sulindac. We conclude that defective FPR1 signaling favors intestinal tumorigenesis through the modulation of the innate inflammatory/immune response.

Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of a novel transcription factor, Musculin, on the biological functions of bone marrow-derived dendritic cells (BMDC) in mice. Methods Bone marrow cells from wildtype (WT) and Musculin knockout (KO, -/-) mice were obtained and cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 so as to induce BMDC in vitro. After 7 d, the BMDC were added with lipopolysaccharide (LPS) and continued to culture for 24 h. Besides the cellular morphological changes were observed dynamically, BMDC and the supernatants were collected from the WT and Musculin-/-cells. Flow cytometry was used to analyze the proportion of positive cells of CD11c, CD40, CD80, CD86, PD-L1 and IAIE in each group. CCK-8 assay was adopted to detect the cellular proliferation for stimulating heterologous lymphocytes and the different stimulation indexes (SI) were calculated respectively. ELISA was applied to detect the supernatant contents of IL-1β, IL-6, IL-10, IL-12 (P40), IL-12 (P70) and TNF-α in each group. Results The BMDC from the Musculin-/-group had similar morphology and no significant difference in purity, decreased percentages of co-stimulatory molecules such as CD40, CD80 and CD86 (P < 0.05), and reduced percentages of co-inhibitory molecules PD-L1 and major histocompatibility complex class Ⅱ (MHC-Ⅱ) IAIE though no significant differences when compared with those of WT group. Moreover, the number of heterologous lymphocytes and related SI after of mixed lymphocyte reaction (MLR) were obvious higher in the Musculin-/-group than the WT group, especially after 5 days' stimulation (P < 0.05). In addition, the supernatant levels of proinflammatory factors including IL-1β, IL-6, IL-12 (P40), IL-12 (P70) and TNF-α were significantly increased in the Musculin-/-group than the WT group (P < 0.05), but no such changes were observed in anti-inflammatory factor IL-10 level between the 2 groups. Conclusion Musculin may enhance the immune function mediated by BMDC and suppress inflammatory reaction, suggesting that it can regulate the biological functions on BMDC.

Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution.

Formyl peptide receptor-1 (FPR1) is a pattern recognition receptor that is mostly expressed by myeloid cells. In patients with colorectal cancer (CRC), a loss-of-function polymorphism (rs867228) in the gene coding for FPR1 has been associated with reduced responses to chemotherapy or chemoradiotherapy. Moreover, rs867228 is associated with accelerated esophageal and colorectal carcinogenesis. Here, we show that dendritic cells from Fpr1−/− mice exhibit reduced migration in response to chemotherapy-treated CRC cells. Moreover, Fpr1−/− mice are particularly susceptible to chronic ulcerative colitis and colorectal oncogenesis induced by the mutagen azoxymethane followed by oral dextran sodium sulfate, a detergent that induces colitis. These experiments were performed after initial co-housing of Fpr1−/− mice and wild-type controls, precluding major Fpr1-driven differences in the microbiota. Pharmacological inhibition of Fpr1 by cyclosporin H also tended to increase intestinal oncogenesis in mice bearing the ApcMin mutation, and this effect was reversed by the anti-inflammatory drug sulindac. We conclude that defective FPR1 signaling favors intestinal tumorigenesis through the modulation of the innate inflammatory/immune response. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) undergo glycolytic reprogramming, a metabolic conversion process essential for their activation. Vitamin D has been reported to affect the function of DCs, but studies in metabolic diseases are insufficient. This study investigates the effects of in vitro 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3) treatment on glycolytic reprogramming of bone marrow-derived dendritic cells (BMDCs) from control, obese, and atherosclerosis mice. Six-week-old male C57BL/6J mice were fed a control diet (CON) or a Western diet (WD), and B6.129S7- Ldlr tm1Her /J mice were fed a Western diet (LDLR−/−) for 16 weeks. BMDCs were cultured in a medium containing 1,25(OH) 2 D 3 (10 nM) for 7 days and stimulated with lipopolysaccharide (LPS, 50 ng/mL) for 24 h. In mature BMDCs, 1,25(OH) 2 D 3 treatment decreased basal and compensatory glycolytic proton efflux rates (glycoPER), the expression of surface markers related to immune function of DCs (MHC class II, CD80, and CD86), and IL-12p70 production. In addition, mTORC1 activation and nitric oxide (NO) production were suppressed by 1,25(OH) 2 D 3 treatment in mature BMDCs. The effect of 1,25(OH) 2 D 3 treatment on IL-12p70 production and mTORC1 activity in the LDLR−/− group was greater than in the CON group. These findings suggest that vitamin D can affect the metabolic environment of BMDCs by regulating glycolytic reprogramming as well as by inducing tolerogenic phenotypes of DCs. [Display omitted] • Metabolism of mature BMDCs is characterized by an increased glycolytic flux. • Vitamin D treatment suppressed glycolysis rates and NO production of mature BMDCs. • The phenotype associated with mature BMDCs was altered by vitamin D treatment. • The effect of vitamin D on IL-12p70 production differed in hyperlipidemic state. • Vitamin D can affect the activation of BMDCs by regulating glycolytic reprogramming. [ABSTRACT FROM AUTHOR]

Purpose: The side effects of immune suppressants on immune rejection have become increasingly apparent after keratoplasty. To find out new alternative immunotherapy strategies, we studied the role of programmed death-1 (PD-1) and its ligand (PD-L1) co-stimulatory pathway in inducing immune tolerance of rat keratoplasty. Methods: The PD-L1 protein was constitutively overexpressed via lentiviral transduction in bone marrow-derived dendritic cells (BMDCs) from rats, then infused via the tail vein into rats before undergoing keratoplasty. Western blot analysis of PD-L1 protein confirmed the effectiveness of lentivirus-mediated. The phenotype of immature BMDC was confirmed by flow cytometry analysis with CD80, CD86, CD11c and MHC-II antibodies. To investigate the mechanism of the immune tolerance induced by BMDCs transfusion, PD-L1, IFN-γ and IL-17 in serum and cell culture supernatant were assessed by ELISA and qPCR. Results: After LPS stimulation, immature dendritic cells with over-expression of PD-L1 still showed high expression of PD-L1(p < 0.001), and low expression of IL-17 and IFN-γ (p < 0.001), which reduced neovascularization (p < 0.05), and prolonged the survival after corneal implants. Conclusion: Immature DC cells with overexpression of PD-L1 have low ability to activate T cells, which is a potential treatment for avoiding graft rejection by promoting natural immunosuppression. This cellular treatment is expected to reduce the use of immune suppressants and the occurrence of side effects. [ABSTRACT FROM AUTHOR]

Arthritis is a disorder that is characterized by joint inflammation and other symptoms. Rheumatoid arthritis (RA), an autoimmune disease, is one of the most common arthritis in worldwide. Inflammation of the synovium is the main factor that triggers bone erosion in the joints in RA, but the pathogenesis of RA is not clearly understood. Kefir grain-fermented products have been demonstrated to enhance immune function and exhibit immune-modulating bioactivities. This study aims to explore the role of kefir peptides (KPs) on the regulation of dendritic cell, which are found in RA synovial fluid, and the protection effects of KPs on mice with collagen-induced arthritis (CIA). Immature mouse bone marrow-derived dendritic cells (BMDCs) were treated with KPs (2.2 and 4.4 mg/ml) and then exposed to lipopolysaccharide (LPS) to study the immune regulation function of KPs in dendritic cells. Mice with CIA (n = 5 per group) were orally administrated KPs (3.75 and 7.5 mg/day/kg) for 21 days and therapeutic effect of KPs on mice with arthritis were assessed. In this study, we found that KPs could inhibit surface molecule expression, reduce inflammatory cytokine release, and repress NF-κB and MAPK signaling in LPS-stimulated mouse BMDCs. In addition, a high dose of KPs (7.5 mg/kg) significantly alleviated arthritis symptoms, decreased inflammatory cytokine expression, suppressed splenic DC maturation and decrease the percentage of Th1 and Th17 in the spleens on mice with CIA. Our findings demonstrated that KPs ameliorate CIA in mice through the mechanism of suppressing DC maturation and inflammatory cytokine releases. [ABSTRACT FROM AUTHOR]

Arthritis is a disorder that is characterized by joint inflammation and other symptoms. Rheumatoid arthritis (RA), an autoimmune disease, is one of the most common arthritis in worldwide. Inflammation of the synovium is the main factor that triggers bone erosion in the joints in RA, but the pathogenesis of RA is not clearly understood. Kefir grain-fermented products have been demonstrated to enhance immune function and exhibit immune-modulating bioactivities. This study aims to explore the role of kefir peptides (KPs) on the regulation of dendritic cell, which are found in RA synovial fluid, and the protection effects of KPs on mice with collagen-induced arthritis (CIA). Immature mouse bone marrow-derived dendritic cells (BMDCs) were treated with KPs (2.2 and 4.4 mg/ml) and then exposed to lipopolysaccharide (LPS) to study the immune regulation function of KPs in dendritic cells. Mice with CIA (n = 5 per group) were orally administrated KPs (3.75 and 7.5 mg/day/kg) for 21 days and therapeutic effect of KPs on mice with arthritis were assessed. In this study, we found that KPs could inhibit surface molecule expression, reduce inflammatory cytokine release, and repress NF-κB and MAPK signaling in LPS-stimulated mouse BMDCs. In addition, a high dose of KPs (7.5 mg/kg) significantly alleviated arthritis symptoms, decreased inflammatory cytokine expression, suppressed splenic DC maturation and decrease the percentage of Th1 and Th17 in the spleens on mice with CIA. Our findings demonstrated that KPs ameliorate CIA in mice through the mechanism of suppressing DC maturation and inflammatory cytokine releases.

BackgroundPrevious studies have shown that lipopolysaccharide (LPS)-activated bone marrow-derived dendritic cells (DClps) might induce tolerance in autoimmune and cancer models in vivo, whereas it remains unclear whether DClps could play a role in allergic disease model. Herein, we aimed to elucidate the potential effects of DClps on OVA-sensitized/challenged airway inflammation in a mouse model, which may help facilitate the application of specific tolerogenic dendritic cells (tolDC) in allergic asthma in the future.MethodsThe phenotype and function of immature DC (DCia), DClps or IL-10-activated-DC (DC10) were determined. OVA-sensitized/challenged mice were treated with OVA-pulsed DCia or DClps or DC10. We assessed the changes of histopathology, serum total IgE level, pulmonary signal transducers and activators of transcription (STAT), pulmonary regulatory T cells (Tregs), and airway recall responses to OVA rechallenge, including proliferation and cytokine secretory function of pulmonary memory CD4+ T cells in the treated mice.ResultsDClps exhibited low levels of CD80 and MHCII and increased levels of anti-inflammatory cytokines such as IL-10 and TGF-β. Additionally, DClps treatment dramatically diminished infiltration of inflammatory cells, eosinophilia, serum IgE and STAT6 phosphorylation level, increased the number of pulmonary Tregs. In addition, DClps treatment decreased the proliferation of pulmonary memory CD4+ T cells, which further rendered the downregulation of Th2 cytokines in vitro.ConclusionLPS stimulation may lead to a tolerogenic phenotype on DC, and thereby alleviated the Th2 immune response of asthmatic mice, possibly by secreting anti-inflammatory cytokines, inhibiting pulmonary memory CD4+ T cells, downregulating pulmonary STAT6 phosphorylation level and increasing pulmonary Tregs.

Background: Previous studies have shown that lipopolysaccharide (LPS)-activated bone marrow-derived dendritic cells (DClps) might induce tolerance in autoimmune and cancer models in vivo , whereas it remains unclear whether DClps could play a role in allergic disease model. Herein, we aimed to elucidate the potential effects of DClps on OVA-sensitized/challenged airway inflammation in a mouse model, which may help facilitate the application of specific tolerogenic dendritic cells (tolDC) in allergic asthma in the future. Methods: The phenotype and function of immature DC (DCia), DClps or IL-10-activated-DC (DC10) were determined. OVA-sensitized/challenged mice were treated with OVA-pulsed DCia or DClps or DC10. We assessed the changes of histopathology, serum total IgE level, pulmonary signal transducers and activators of transcription (STAT), pulmonary regulatory T cells (Tregs), and airway recall responses to OVA rechallenge, including proliferation and cytokine secretory function of pulmonary memory CD4+ T cells in the treated mice. Results: DClps exhibited low levels of CD80 and MHCII and increased levels of anti-inflammatory cytokines such as IL-10 and TGF-β. Additionally, DClps treatment dramatically diminished infiltration of inflammatory cells, eosinophilia, serum IgE and STAT6 phosphorylation level, increased the number of pulmonary Tregs. In addition, DClps treatment decreased the proliferation of pulmonary memory CD4+ T cells, which further rendered the downregulation of Th2 cytokines in vitro. Conclusion: LPS stimulation may lead to a tolerogenic phenotype on DC, and thereby alleviated the Th2 immune response of asthmatic mice, possibly by secreting anti-inflammatory cytokines, inhibiting pulmonary memory CD4+ T cells, downregulating pulmonary STAT6 phosphorylation level and increasing pulmonary Tregs. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNP) are one of the most studied nanoparticles due to their anti-bacterial, -fungal, -viral, -parasitic, and -inflammatory properties. This raises the need to evaluate the toxicity and biological effects of AgNP in the immune system in order to develop new safer biomedical products. In this study, an AgNP formulation currently approved for veterinary applications was applied to mouse bone marrow-derived dendritic cells (BMDC), considered important antigen-presenting cells of the immune system, to evaluate cytotoxicity, genotoxicity, and any significant influence on expression of cellular markers associated with BMDC phenotype and maturation status. The results showed that after 12 h of AgNP exposure, a significant decrease in BMDC viability occurred at the highest concentration tested (1.0 µg AgNP/ml) and at lower doses, the cells maintained membrane integrity and metabolic activity. DNA damage was not significant with any AgNP level aside from the 1.0 µg AgNP/ml level. Regarding phenotype, no differences in expression of CD40 (co-stimulatory molecule highly present in mature BMDC) or in CD273 (a marker for inhibitory T-cell response) were observed. The current results showed that the toxicity of this AgNP formulation was dose-related. The findings also suggest BMDC could maintain structural conservation of co-stimulatory/co-inhibitory surface molecules after 12 h of exposure to this AgNP. This work represents the first step in identifying the toxic effects of this AgNP formulation on dendritic cells.

B-cell activating factor (BAFF) plays a crucial role in survival, differentiation, and antibody secretion of B cells. Microbial products with B-cell mitogenic properties can indirectly promote expansion and activation of B cells by stimulating accessory cells, such as dendritic cells (DCs), to induce BAFF. Although bacterial lipoproteins are potent B-cell mitogen like lipopolysaccharides (LPSs), it is uncertain whether they can stimulate DCs to induce BAFF expression. Here, we evaluated the effect of bacterial lipoproteins on BAFF expression in mouse bone marrow–derived DCs. Lipoprotein-deficient Staphylococcus aureus mutant induced relatively low expression level of membrane-bound BAFF (mBAFF) and the mRNA compared with its wild-type strain, implying that bacterial lipoproteins can positively regulate BAFF induction. The synthetic lipopeptides Pam2CSK4 and Pam3CSK4, which mimic bacterial lipoproteins, dose-dependently induced BAFF expression, and their BAFF-inducing capacities were comparable to those of LPS in DCs. Induction of BAFF by the lipopeptide was higher than the induction by other microbe-associated molecular patterns, including peptidoglycan, flagellin, zymosan, lipoteichoic acid, and poly(I:C). Pam3CSK4 induced both mBAFF and soluble BAFF expression in a dose- and time-dependent manner. BAFF expression by Pam3CSK4 was completely absent in DCs from TLR2- or MyD88-deficient mice. Among various MAP kinase inhibitors, only JNK inhibitors blocked Pam3CSK4-induced BAFF mRNA expression, while inhibitors blocking ERK or p38 kinase had no such effect. Furthermore, Pam3CSK4 increased the DNA-binding activities of NF-κB and Sp1, but not that of C/EBP. Pam3CSK4-induced BAFF promoter activity via TLR2/1 was blocked by NF-κB or Sp1 inhibitor. Collectively, these results suggest that bacterial lipoproteins induce expression of BAFF through TLR2/MyD88/JNK signaling pathways leading to NF-κB and Sp1 activation in DCs, and BAFF derived from bacterial lipoprotein-stimulated DCs induces B-cell proliferation.

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types.

B-cell activating factor (BAFF) plays a crucial role in survival, differentiation, and antibody secretion of B cells. Microbial products with B-cell mitogenic properties can indirectly promote expansion and activation of B cells by stimulating accessory cells, such as dendritic cells (DCs), to induce BAFF. Although bacterial lipoproteins are potent B-cell mitogen like lipopolysaccharides (LPSs), it is uncertain whether they can stimulate DCs to induce BAFF expression. Here, we evaluated the effect of bacterial lipoproteins on BAFF expression in mouse bone marrow–derived DCs. Lipoprotein-deficient Staphylococcus aureus mutant induced relatively low expression level of membrane-bound BAFF (mBAFF) and the mRNA compared with its wild-type strain, implying that bacterial lipoproteins can positively regulate BAFF induction. The synthetic lipopeptides Pam2CSK4 and Pam3CSK4, which mimic bacterial lipoproteins, dose-dependently induced BAFF expression, and their BAFF-inducing capacities were comparable to those of LPS in DCs. Induction of BAFF by the lipopeptide was higher than the induction by other microbe-associated molecular patterns, including peptidoglycan, flagellin, zymosan, lipoteichoic acid, and poly(I:C). Pam3CSK4 induced both mBAFF and soluble BAFF expression in a dose- and time-dependent manner. BAFF expression by Pam3CSK4 was completely absent in DCs from TLR2- or MyD88-deficient mice. Among various MAP kinase inhibitors, only JNK inhibitors blocked Pam3CSK4-induced BAFF mRNA expression, while inhibitors blocking ERK or p38 kinase had no such effect. Furthermore, Pam3CSK4 increased the DNA-binding activities of NF-κB and Sp1, but not that of C/EBP. Pam3CSK4-induced BAFF promoter activity via TLR2/1 was blocked by NF-κB or Sp1 inhibitor. Collectively, these results suggest that bacterial lipoproteins induce expression of BAFF through TLR2/MyD88/JNK signaling pathways leading to NF-κB and Sp1 activation in DCs, and BAFF derived from bacterial lipoprotein-stimulated DCs induces B-cell proliferation. [ABSTRACT FROM AUTHOR]

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNP) are one of the most studied nanoparticles due to their anti-bacterial, -fungal, -viral, -parasitic, and -inflammatory properties. This raises the need to evaluate the toxicity and biological effects of AgNP in the immune system in order to develop new safer biomedical products. In this study, an AgNP formulation currently approved for veterinary applications was applied to mouse bone marrow-derived dendritic cells (BMDC), considered important antigen-presenting cells of the immune system, to evaluate cytotoxicity, genotoxicity, and any significant influence on expression of cellular markers associated with BMDC phenotype and maturation status. The results showed that after 12 h of AgNP exposure, a significant decrease in BMDC viability occurred at the highest concentration tested (1.0 µg AgNP/ml) and at lower doses, the cells maintained membrane integrity and metabolic activity. DNA damage was not significant with any AgNP level aside from the 1.0 µg AgNP/ml level. Regarding phenotype, no differences in expression of CD40 (co-stimulatory molecule highly present in mature BMDC) or in CD273 (a marker for inhibitory T-cell response) were observed. The current results showed that the toxicity of this AgNP formulation was dose-related. The findings also suggest BMDC could maintain structural conservation of co-stimulatory/co-inhibitory surface molecules after 12 h of exposure to this AgNP. This work represents the first step in identifying the toxic effects of this AgNP formulation on dendritic cells. [ABSTRACT FROM AUTHOR]

Background: Peste des petits ruminants virus (PPRV) is the causative agent of PPR, which can cause an acute, highly contagious and fatal disease of sheep and goats, resulting in significant economic losses for commercial animal husbandry due to its high mortality and morbidity. As professional antigen-presenting cells, dendritic cells (DCs) play a unique role in innate immunity. This study aimed to gain a deeper understanding of the transcriptional response of bone marrow-derived dendritic cells (BMDCs) stimulated with PPRV. Results: Transcriptional profiling was performed using RNA sequencing. Herein, we reported that compared to untreatedBMDCs, 4492 differentially expressed genes (DEGs) were identified following PPRV stimulation, out of these DEGs 2311 were upregulated and 2181 were downregulated, respectively. A total of three gene ontology (GO) term clusters of biological process, cell component and molecular function were significantly enriched in 963GOterms in the PPRV-stimulated BMDCs. TheseGOclusters were related to inflammatory response, cell division and vacuole, anchoring junction, positive regulation of cellular component and nucleoside binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs were enriched in a chemokine signaling pathway, protein processing in endoplasmic reticulum, cell cycle and mTOR signaling pathway. Additionally, identified DEGs of BMDCs were further validated by qRT-PCR and the results were in accordance with the change of the genes. This study suggested the effects of PPRV stimulation on the maturation and function of BMDCs. Conclusion: We found that the dramatic BMDCs transcriptome changes triggered were predominantly related to an inflammatory response and chemokine signaling pathway. [ABSTRACT FROM AUTHOR]

Extra virgin olive oil (EVOO) represents one of the most important health-promoting foods whose antioxidant and anti-inflammatory activities are mainly associated to its polyphenols content. To date, studies exploring the effect of EVOO polyphenols on dendritic cells (DCs), acting as a crosstalk between the innate and the adaptive immune response, are scanty. Therefore, we studied the ability of three EVOO extracts (cv. Coratina, Cima di Mola/Coratina, and Casaliva), characterized by different polyphenols amount, to regulate DCs maturation in resting conditions or after an inflammatory stimulus. Cima di Mola/Coratina and Casaliva extracts were demonstrated to be the most effective in modulating DCs toward an anti-inflammatory profile by reduction of TNF and IL-6 secretion and CD86 expression, along with a down-modulation of Il-1β and iNOS expression. From factorial analysis results, 9 polyphenols were tentatively established to play a synergistic role in modulating DCs inflammatory ability, thus reducing the risk of chronic inflammation.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most common diseases in the global swine industry. PRRSV infection is highly restricted to cells of the monocyte-macrophage lineage. However, the lack of antibodies to swine monocyte-macrophage lineage markers significantly hampers PRRSV research. In this study, we have developed a monoclonal antibody against the swine leukocyte antigen (SLA)-DRα chain and confirmed its reactivity with endogenous expressed SLA-DR in a variety of cell lines and primary swine antigen-presenting cells (PAMs, PBMC and BM-DCs). Moreover, the level of SLA-DR expression after PRRSV infection were evaluated by our homemade Mab and a commercial anti-SLA-DR antibody. Based on our result, the protein level of SLA-DRα expression is increased after PRRSV infection in DC, while the mRNA of both SLA-DRα and SLA-DRβ were significantly inhibited by PRRSV replication. In conclusion, we successfully developed a MAb reactive with endogenous SLA-DR in western blotting, and this MAb could be a useful tool for further research and analysis. Moreover, the inconsistency of SLA-DR expression between protein and mRNA levels may suggest a novel role of DC played during the immune response after PRRSV infection. [ABSTRACT FROM AUTHOR]

Pathogen recognition receptor (PRR) signaling is critical for triggering innate immune activation and the expression of immune response genes, including genes that impart restriction against virus replication. RIG‐I‐like receptors and TLRs are PRRs that signal immune activation and drive the expression of antiviral genes and the production of type I IFN leading to induction of IFN‐stimulated genes, in part through the interferon regulatory factor (IRF) family of transcription factors. Previous studies with West Nile virus (WNV) showed that IRF3 and IRF7 regulate IFN expression in fibroblasts and neurons, whereas macrophages and dendritic cells (DCs) retained the ability to induce IFN‐β in the absence of IRF3 and IRF7 in a manner implicating IRF5 in PRR signaling actions. Here we assessed the contribution of IRF5 to immune gene induction in response to WNV infection in DCs. We examined IRF5‐dependent gene expression and found that loss of IRF5 in mice resulted in modest and subtle changes in the expression of WNV‐regulated genes. Anti‐IRF5 chromatin immunoprecipitation with next‐generation sequencing of genomic DNA coupled with mRNA analysis revealed unique IRF5 binding motifs within the mouse genome that are distinct from the canonical IRF binding motif and that link with IRF5‐target gene expression. Using integrative bioinformatics analyses, we identified new IRF5 primary target genes in DCs in response to virus infection. This study provides novel insights into the distinct and unique innate immune and immune gene regulatory program directed by IRF5. IRF5 regulates distinct gene regulation programs associated with diverse cellular pathways following West Nile Virus infection in mouse dendritic cells.. [ABSTRACT FROM AUTHOR]

Abstract The immunoregulation and immunopotentiation of Polysaccharides of Atractylodes macrocephala Koidz (PAMK) have been widely demonstrated. Nanostructured lipid carriers (NLC) have high drug loading capacity for lipophilic and hydrophilic drugs, and have good biocompatibility and high bioavailability. In this study, the effect of PAKM-NLC on the surface molecule expression of bone marrow-derived dendritic cells (BMDCs) in vitro was investigated by flow cytometry, and the cytokines secreted by dendritic cell supernatants were detected by ELISA. The results showed that compared with other control groups, PAMK-NLC could significantly increase the expression of CD80 and CD86 and promote the secretion of IL-1β, IL-12, TNF-α and IFN-γ, indicating that PAMK-NLC have a more pronounced effect on the maturation and differentiation of BMDCs. In addition, effects of PAMK-NLC nanoparticles on OVA-immunized mice were explored. Compared with other control groups, PAMK-NLC-OVA can significantly promote the production of OVA-specific antibodies in serum, stimulate the secretion of cytokines, increase the proliferation rate of spleen lymphocytes after OVA re-stimulation, and induce stronger activation of CD3+CD4+ and CD3+CD8+ lymphocytes. As an adjuvant of OVA, PAMK-NLC has a better immunological enhancement effect than PAMK or blank NLC, and has good adjuvant activity. [ABSTRACT FROM AUTHOR]

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson’s disease. Although the characteristics of LRRK2 have gradually been revealed, its true physiological functions remain unknown. LRRK2 is highly expressed in immune cells such as B2 cells and macrophages, suggesting that it plays important roles in the immune system. In the present study, we investigate the roles of LRRK2 in the immune functions of dendritic cells (DCs). Bone marrow-derived DCs from both C57BL/6 wild-type (WT) and LRRK2 knockout (KO) mice were induced by culture with granulocyte/macrophage-colony stimulating factor (GM/CSF) in vitro. We observed the differentiation of DCs, the phosphorylation of the transcriptional factors NF-κB, Erk1/2, and p-38 after lipopolysaccharide (LPS) stimulation and antigen-presenting ability by flow cytometry. We also analyzed the production of inflammatory cytokines by ELISA. During the observation period, there was no difference in DC differentiation between WT and LRRK2-KO mice. After LPS stimulation, phosphorylation of NF-κB was significantly increased in DCs from the KO mice. Large amounts of inflammatory cytokines were produced by DCs from KO mice after both stimulation with LPS and infection with Leishmania. CD4+ T-cells isolated from antigen-immunized mice proliferated to a significantly greater degree upon coculture with antigen-stimulated DCs from KO mice than upon coculture with DCs from WT mice. These results suggest that LRRK2 may play important roles in signal transduction and antigen presentation by DCs.

As a kind of superantigen, staphylococcal enterotoxin C2 (SEC2) is well known as a powerful immunomodulator. However, most previous studies about SEC2 focus on its T cell activating characters. But the direct effect of SEC2 on antigen-presenting cells (APCs) which are important for the T cell activation is not clearly. In this study, we investigated the effect of SEC2 on murine bone marrow-derived dendritic cells (BMDCs) which are known as the specialized professional APCs. Contrary to its effects on T cells, SEC2 could not induce proliferation or cytotoxicity to BMDCs even in high concentrations. While SEC2 could promote the mature of BMDCs with increased expression of co-stimulatory molecules on cell membrane such as CD80, CD86, and MHC II. The production of pro-inflammatory cytokines such as TNF- α , IFN-γ and IL-6 were also increased in BMDCs treated with SEC2. We also found that SEC2 enhanced the genes expression of pattern recognition receptors including toll-like receptors 2 (TLR2) and TLR4 in BMDCs, and up-regulated the key signal molecule MyD88 in both mRNA and protein levels. In addition, SEC2 also caused IκB α degradating and NFκB p65 translocating from the cytoplasm to the nucleus in BMDCs. The siRNAs for both TLR2 and TLR4, as well as NFκB specific inhibitor BAY 11–7085 could inhibit the co-stimulatory molecules expression and pro-inflammatory cytokines releasing induced by SEC2. Moreover, TLR2/4 specific siRNAs inhibited p65 and MyD88 upregulation induced by SEC2. In summary, all our results indicated that SEC2 could stimulate BMDCs maturation through TLR-NFκB signaling pathways. [ABSTRACT FROM AUTHOR]

Highlights • Immature BMDC were more efficient than mature BMDC in supporting PRRSV1 replication. • PRRSV1 was seen attached to BMDC even in absence of CD163, PoSn or heparan sulphate. • PRRSV1 infected CD163− BMDC only when CD163low/high subsets were co-cultured. Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect porcine dendritic cells (DC). Previous studies indicated that different PRRSV1 isolates regulated differently the cytokine profiles and expression of surface molecules of DC. However, the characterisation of the infection is lacking. The current study aimed to characterise the replication and attachment of different PRRSV1 isolates in bone marrow-derived DC (BMDC). For this purpose, immature (i) and mature (m) BMDC were infected with three PRRSV1 isolates. The replication kinetics showed that titres in iBMDC were significantly ( p < 0.05) higher than in mBMDC by 24 hpi, and for two isolates titres peaked earlier in iBMDC, suggesting that iBMDC were more efficient in supporting PRRSV1 replication than mBMDC. The attachment was revealed by a three-colour confocal microscopy staining. All three isolates were seen attached to iBMDC even in cells lacking CD163 -the essential receptor for PRRSV- or porcine sialoadhesin (PoSn). The attachment was not fully avoided after removal of heparan sulphate by heparinase I. Furthermore, the infection was examined with regards to CD163 expression. By flow cytometry and confocal microscopy, positive signals of PRRSV1 nucleocapsid could be observed in CD163− iBMDC. Additional sorting experiment demonstrated that CD163− iBMDC were infected only when CD163lo/hi cells were present. This can be interpreted in different ways: susceptible CD163− cells arose as result of milieu created by CD163+ infected BMDC; CD163− cells were infected by receptor-independent mechanisms ( i.e. exosomes) or, some cells expressed CD163 at levels beyond the technical sensitivity. [ABSTRACT FROM AUTHOR]

Certain food components possess immunomodulatory effects. The aim of this study was to elucidate the mechanism of the immunostimulatory activity of Brassica rapa L. We demonstrated an enhancement of natural killer (NK) activity and interferon (IFN)-γ production in mice that were orally administered an insoluble fraction of B. rapa L. The insoluble fraction of B. rapa L. significantly induced IFN-γ production in mouse spleen cells in an interleukin (IL)-12-dependent manner, and NK1.1+ cells were the main cells responsible for producing IFN-γ. Additionally, the results suggested that the active compounds in the insoluble fraction were recognized by Toll-like receptor (TLR) 2, TLR4, and C-type lectin receptors on dendritic cells, and they activated signaling cascades such as MAPK, NF-κB, and Syk. These findings suggest that B. rapa L. is a potentially promising immuno-improving material, and it might be useful for preventing immunological disorders such as infections and cancers by activating innate immunity. [ABSTRACT FROM AUTHOR]

Cytokine induction is often critical for the host defense during acute immune responses while, if not tightly regulated, it may cause an immunological pathology coincident with tissue damage. Despite the fact that gram-positive bacterial infection has become increasingly prevalent, immune modulation induced by lipoteichoic acid (LTA), the major cell wall component of gram-positive bacteria has not been studied thoroughly at the cellular level. In the current study, tolerance induction in mouse bone marrow-derived dendritic cells (BMDCs) treated with single or repeated stimulation of Staphylococcus aureus LTA was compared with those of Escherichia coli lipopolysaccharide (LPS). The results showed that repeated LTA stimulation significantly suppressed pro-inflammatory cytokine (TNF-α and IL-6) production in BMDCs, comparable to that of LPS, but with less extent, down-regulated IL-10 and enhanced the inhibitory molecule, LAG-3-associated protein (LAP). Furthermore, we observed a sustained expression of unique negative regulators, Toll interacting protein (TOLLIP) and Indoleamine 2,3-dioxygenase (IDO), in BMDCs treated with LTA. A transient hyporesponsiveness period appeared when DCs were treated repeatedly with LTA or LPS showing a distinctive pattern. Intriguingly, LPS exposure induced cross tolerance to LTA while LTA exposure did not to LPS, implicating that a distinct signaling components are involved in response to LTA. Collectively, a distinct immune regulation appeared to be responsible for the LPS- and LTA-induced tolerance on cytokine production, expression of surface markers and intracellular proteins. [ABSTRACT FROM AUTHOR]

Background Identification of the allergenic potency of chemicals is a key step in the safety assessment process. Predictive assays that require no or few animals are needed. Objectives To develop an alternative in vitro mouse bone marrow-derived dendritic cell ( BMDC) assay to determine the allergenic potential of chemicals. Methods BMDCs were exposed to well-known allergens and to non-allergenic chemicals. Surface marker expression and cytokine release of BMDCs were analysed after treatment. Results Eleven tested chemicals showed a significant stimulation index ( SI) of >1.5 (accuracy, 75%; sensitivity, 69%). The four non-allergens all showed a SI of <1.5. Eight contact allergens tested showed a significant SI of >1.5 (accuracy, 92%; sensitivity, 89%), whereas only two respiratory allergens showed a significant SI of >1.5 (accuracy, 60%; sensitivity, 33%). Conclusions The results indicate that the BMDC assay could become a reliable test for assessment of the allergenic potential of chemicals. The next step should include the testing of further chemicals, with the aim of integrating this assay into the toolbox of in vitro methods for the evaluation of the allergenic potential of chemicals. [ABSTRACT FROM AUTHOR]

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity. [ABSTRACT FROM AUTHOR]

Activated dendritic cells (DCs) undergo significant metabolic reprogramming, which is characterized by an increase in aerobic glycolysis and a concurrent progressive loss of oxidative phosphorylation. The modulation of metabolic reprogramming is believed to be closely related to the function of DCs. Vitamin D has been reported to inhibit the maturation of DCs. DC dysfunction has been reported in diabetic patients, and hyperglycemia is associated with impaired glycolytic metabolism in immune cells. Therefore, vitamin D and diabetes may affect intracellular metabolism, thereby regulating the activity of DCs. We investigated the effect of in vitro treatment of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3) on metabolic reprogramming and maturation of bone marrow-derived dendritic cells (BMDCs) from diabetic mouse. Six-week-old male C57BLKS/J-m+/m+ mice (CON) and C57BLKS/J-db/db mice (db/db) were fed with a 10% kcal fat diet for seven weeks. BMDCs were generated by culturing bone marrow cells from the mice with rmGM-CSF (20 ng/mL) in the absence or presence of 10 nM 1,25(OH) 2 D 3. The maturation of BMDCs was induced via lipopolysaccharide (LPS, 50 ng/mL) stimulation for 24 h. LPS stimulation induced iNOS protein expression and decreased the mitochondrial respiration, while increased lactate production and the expression of glycolytic pathway-related genes (Glut1 and Pfkfb3) in BMDCs from both CON and db/db groups. In LPS-stimulated mature BMDCs, 1,25(OH) 2 D 3 treatment decreased the expression of surface markers related to immunostimulatory functions (MHC class II, CD80, CD86, and CD40) and production of IL-12p70 in both CON and db/db groups. While the mRNA level of the gene related to glucose uptake (Glut1) was increased in both groups, lactate production was decreased by 1,25(OH) 2 D 3 treatment. mTORC1 activity was suppressed following 1,25(OH) 2 D 3 treatment. Collectively, our findings confirmed that metabolic reprogramming occurred in BMDCs following LPS stimulation. In vitro 1,25(OH) 2 D 3 treatment induced tolerogenic phenotypes by reducing the expression of surface markers, as well as cytokine production. However, no significant difference was observed regarding the effects of 1,25(OH) 2 D 3 treatment on metabolic conversion and maturation of BMDCs between the control and diabetic mice. Additionally, the decreased aerobic glycolysis induced by the 1,25(OH) 2 D 3 treatment appeared to be associated with the diminished maturation of BMDCs, and mTORC1 appears to play a key role in the 1,25(OH) 2 D 3 -mediated regulation of glycolysis. • Metabolic reprogramming occurred in BMDCs following LPS stimulation. • Vitamin D treatment induces semi-mature phenotypes of BMDCs. • Decreased glycolysis induced by vitamin D treatment appeared to affect BMDCs' maturation. • Diabetes did not affect the maturation and metabolic reprogramming of BMDCs. • mTORC1 appears to play a key role in the vitamin D -mediated regulation of glycolysis [ABSTRACT FROM AUTHOR]

Imperatorin is a furanocoumarin compound which exists in many medicinal herbs and possesses various biological activities. Herein, we investigated the antiallergic effects of imperatorin in asthmatic mice and explored the immunomodulatory actions of imperatorin on immune cells. We used a murine model of ovalbumin (OVA)-induced asthma to evaluate the therapeutic potential of imperatorin. Additionally, bone marrow-derived dendritic cells (DCs; BMDCs) were used to clarify whether imperatorin exerts an antiallergic effect through altering the ability of DCs to regulate T cells. Oral administration of imperatorin to OVA-sensitized and −challenged mice decreased serum OVA-specific immunoglobulin E (IgE) production, attenuated the airway hyperresponsiveness (AHR), and alleviated airway inflammation in a dose-dependent manner. Notably, secretions of Th2 cytokines and chemokines were reduced, and numbers of interleukin (IL)-10-producing regulatory T cells (Tregs) increased in imperatorin-treated mice. Imperatorin inhibited proinflammatory cytokines and IL-12 production but enhanced IL-10 secretion by lipopolysaccharide (LPS)-stimulated BMDCs. Compared to fully mature DCs, imperatorin-treated DCs expressed high levels of the inducible costimulatory ligand (ICOSL) and Jagged1 molecules, and had the regulatory capacity to promote the generation of IL-10-producing CD4 + T cells in vitro. Additionally, imperatorin directly suppressed activated CD4 + T-cell proliferation and cytokine production. Imperatorin may possess therapeutic potential against Th2-mediated allergic asthma not only via stimulating DC induction of Tregs but also via direct inhibition of Th2 cell activation. These findings provide new insights into how imperatorin affects the Th2 immune response and the development of imperatorin as a Treg-type immunomodulatory agent to treat allergic asthma. [ABSTRACT FROM AUTHOR]

Background: The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. Results: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4+ and CD8+ cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. Conclusions: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles. [ABSTRACT FROM AUTHOR]

Background: HemoHIM, which is an herbal preparation of three edible herbs (Angelicam gigas Nakai, Cnidium offinale Makino, and Peaonia japonica Miyabe), is known to have various biological and immunological activities, but the modulatory effects of this preparation on dendritic cells (DCs)-mediated immune responses have not been examined previously. DCs are a unique group of white blood cells that initiate primary immune responses by capturing, processing, and presenting antigens to T cells. Results: In the present study, we investigated the effect of HemoHIM on the functional and phenotypic maturation of murine bone marrow-derived dendritic cells (BMDCs) both in vitro and in vivo. The expression of co-stimulatory molecules (CD40, CD80, CD86, MHC I, and MHC II) and the production of cytokines (IL-1β, IL-6, IL-12p70, and TNF-α) were increased by HemoHIM in BMDCs. Furthermore, the antigen-uptake ability of BMDCs was decreased by HemoHIM, and the antigen-presenting ability of HemoHIM-treated mature BMDCs increased TLR4-dependent CD4+ and CD8+ T cell responses. Conclusions: Our findings demonstrated that HemoHIM induces TLR4-mediated BMDCs functional and phenotypic maturation through in vivo and in vitro. And our study showed the antigen-presenting ability that HemoHIM-treated mature BMDCs increase CD4+ and CD8+ T cell responses by in vitro. These results suggest that HemoHIM has the potential to mediate DC immune responses. [ABSTRACT FROM AUTHOR]

Background: Low, noncytotoxic concentrations of various chemotherapeutic drugs like 5-fluorouracil (5-FU) induce antitumor immune responses by selectively depleting tumor-induced immunosuppressive cells, and could therefore be used in combination with dendritic cell (DC) vaccines in order to enhance their immunotherapeutic efficacy. However, the likely negative influences of low, noncytotoxic doses of 5-FU on bone marrow-derived (BM)-DCs in vitro have not yet been investigated. Methods: The effects of low, noncytotoxic concentrations of 5-FU on mouse BM-DC differentiation and maturation markers (CD11c, MHC class II and CD80) as well as antigen-presenting capacity and cytokine production (IL-12p70 and IL-10) have been assessed. Results: Different low doses of 5-FU had no significant effect on the expression of DC differentiation and maturation or on costimulatory markers (p = 0.5). Furthermore, suboptimal doses of 5-FU did not affect the immunostimulatory functions of DCs such as antigen presentation (p = 0.6) and cytokine production (p = 0.9). Conclusions: These data suggest that low doses of 5-FU have no adverse effects on DC maturation and function, and the efficacy of DC-based cancer immunotherapy may be greatly enhanced by combining it with suboptimal doses of 5-FU. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Dendritic cells, the most powerful type of antigen-presenting cells, have the unique ability to induce primary immune responses. Connexin43 expression is upregulated to increase gap junctions when immune cells are exposed to inflammatory factors. The present study applied small-interfering RNA (siRNA) to decrease connexin43 expression. The results showed that silencing of connexin43 using siRNA resulted in arrest of bone marrow-derived dendritic cell (BM-DC) maturation as evidenced by reduced expression of major histocompatibility complex II, CD40, CD80 and CD86. Functionally, connexin43-silenced BM-DC showed a markedly decreased capability to induce T-cell stimulation. In conclusion, the present study demonstrated that antigens present on BM-DCs can be suppressed by connexin43 knockdown in BM-DCs. The present study therefore presented an effective method to modulate the immunology of BM-DCs. [ABSTRACT FROM AUTHOR]

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen-induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis ( AR), we generated bone marrow-derived dendritic cells ( BMDCs), which were pulsed by ovalbumin ( OVA) and α-galactosylceramide ( OVA/α- GalCer- BMDCs) and administered into the oral submucosa of OVA-sensitized mice before nasal challenge. Nasal symptoms, level of OVA-specific immunoglobulin ( IgE), and T helper type 2 ( Th2) cytokine production in cervical lymph nodes ( CLNs) were significantly ameliorated in wild-type ( WT) mice treated with OVA/α- GalCer- BMDCs, but not in WT mice treated with OVA- BMDCs. These anti-allergic effects were not observed in Jα18-/- recipients that lack iNKT cells, even after similar treatment with OVA/α- GalCer- BMDCs in an adoptive transfer study with CD4+ T cells and B cells from OVA-sensitized WT mice. In WT recipients of OVA/α- GalCer- BMDCs, the number of interleukin ( IL)-21-producing iNKT cells increased significantly and the Th1/ Th2 balance shifted towards the Th1 dominant state. Treatment with anti- IL-21 and anti-interferon ( IFN)-γ antibodies abrogated these anti-allergic effects in mice treated with α- GalCer/ OVA- BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti-allergic effects through production of IL-21 or IFN-γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR. [ABSTRACT FROM AUTHOR]

Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell-DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo-repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single-cell level, C57BL/6 NK-cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS-activated mature DCs induced a high level of chemotactic movement of IL-2-activated NK cells in vitro. We confirmed these findings in a standard trans-well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony-stimulating factor in repulsing NK-cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK-cell migratory responses in NK cell-DC crosstalk will provide new insights into the development of DC-based NK-cell therapies against tumor and infections. [ABSTRACT FROM AUTHOR]

In sporotrichosis, dermal dendritic cells were considered to participate in induction of the immune responses against Sporothrix schenckii infection. However, it is still unclear whether and how dermal dendritic cells were involved in the progress. To clarify the pathogenic role of dermal dendritic cells ( DC) in sporotrichosis, we examined the phagocytosis, maturation stages, cytokine production and antigen-presenting ability of mouse bone marrow-derived DC after stimulation with S. schenckii. By analysis of flow cytometry, electron microscope and confocal microscope, mouse bone marrow-derived DC were proved to be able to phagocytize the S. schenckii. The increased expression of CD40, CD80 and CD86 on the surface of S. schenckii-pulsed mouse bone marrow-derived DC was detected by flow cytometer, indicating that the S. schenckii-pulsed mouse bone marrow-derived DC underwent the maturation program. The secretory enhancement of interleukin ( IL)-12, but not IL-4, was found in S. schenckii-pulsed mouse bone marrow-derived DC, suggesting the possible activation of T-helper 1 prone immune responses. Furthermore, S. schenckii-pulsed mouse bone marrow-derived DC were demonstrated to be capable of inducing the proliferation of T lymphocytes from BALB/c mice that were pre-sensitized with S. schenckii. Together, all the results implied that dermal DC may participate in the induction of immune responses against S. schenckii infection in sporotrichosis. [ABSTRACT FROM AUTHOR]