Dendritic Cell IL-12 Research Articles

Defective IL-23/IL-17 Axis Protects p47phox-/- Mice from Colon Cancer.

In the colon, a sophisticated balance between immune reaction and tolerance is absolutely required. Dysfunction may lead to pathologic phenotypes ranging from chronic inflammatory processes to cancer development. Two prominent modulators of colon inflammation are represented by the closely related cytokines interleukin (IL)-12 and IL-23, which initiate adaptive Th1 and Th17 immune responses, respectively. In this study, we investigated the impact of the NADPH oxidase protein p47phox, which negatively regulates IL-12 in dendritic cells, on colon cancer development in a colitis-associated colon cancer model. Initially, we found that IL-12−/− mice developed less severe colitis but are highly susceptible to colon cancer. By contrast, p47phox−/− mice showed lower tumor scores and fewer high grade tumors than wild-type (WT) littermates. Treatment with toll-like receptor 9 ligand CpG2216 significantly enhanced colitis in p47phox−/− mice, whereas tumor growth was simultaneously reduced. In tumor tissue of p47phox−/− mice, the IL-23/IL-17 axis was crucially hampered. IL-23p19 protein expression in tumor tissue correlated with tumor stage. Reconstitution of WT mice with IL-23p19−/− bone marrow protected these mice from colon cancer, whereas transplantation of WT hematopoiesis into IL-23p19−/− mice increased the susceptibility to tumor growth. Our study strengthens the divergent role of IL-12 and IL-23 in colon cancer development. With the characterization of p47phox as a novel modulator of both cytokines our investigation introduces a promising new target for antitumor strategies.

Desirable cytolytic immune effector cell recruitment by interleukin-15 dendritic cells.

Success of dendritic cell (DC) therapy in treating malignancies is depending on the DC capacity to attract immune effector cells, considering their reciprocal crosstalk is partially regulated by cell-contact-dependent mechanisms. Although critical for therapeutic efficacy, immune cell recruitment is a largely overlooked aspect regarding optimization of DC vaccination. In this paper we have made a head-to-head comparison of interleukin (IL)-15-cultured DCs and conventional IL-4-cultured DCs with regard to their proficiency in the recruitment of (innate) immune effector cells. Here, we demonstrate that IL-4 DCs are suboptimal in attracting effector lymphocytes, while IL15 DCs provide a favorable chemokine milieu for recruiting CD8+ T cells, natural killer (NK) cells and gamma delta (γδ) T cells. Gene expression analysis revealed that IL-15 DCs exhibit a high expression of chemokines involved in antitumor immune effector cell attraction, while IL-4 DCs display a more immunoregulatory profile characterized by the expression of Th2 and regulatory T cell-attracting chemokines. This is confirmed by functional data indicating an enhanced recruitment of granzyme B+ effector lymphocytes by IL-15 DCs, as compared to IL-4 DCs, and subsequent superior killing of tumor cells by the migrated lymphocytes. Elevated CCL4 gene expression in IL-15 DCs and lowered CCR5 expression on both migrated γδ T cells and NK cells, led to validation of increased CCL4 secretion by IL15 DCs. Moreover, neutralization of CCR5 prior to migration resulted in an important inhibition of γδ T cell and NK cell recruitment by IL-15 DCs. These findings further underscore the strong immunotherapeutic potential of IL-15 DCs.

TRAF6 regulates the effects of polarized maturation of tolerability: Marrow-derived dendritic cells on collagen-induced arthritis in mice.

The study aimed to investigate the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and a differentially mature dendritic cell (mDC) in collagen-induced arthritis (CIA) mice and to determine whether or not TRAF6 regulates the activation of an immature dendritic cell (iDC) and inhibits iDC maturation to induce immune tolerance. The mouse bone marrow stem cells were induced with recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) to differentiate immature dendritic cells (DCs), which were divided into four groups with different maturation states: rmGM-CSF, rmIL-4; TNF-α; LPS; and FK506 group. The levels of the cell surfaces of CD80, CD86, and MHI-II were analyzed by flow cytometry to prove DCs at different levels of maturity. The expression of IL-12 in DCs at different maturation states was detected by enzyme-linked immunosorbent assay (ELISA). The expression of TRAF6 mRNA and protein in each group of DCs was detected by a reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The results revealed that the differentiation of bone marrow cells into iDCs was significantly induced by cytokines (rmGM-CSF, IL-4). CD80, CD86, MHC-II were expressed in the four groups, and the difference between them was statistically significant (P<0.05). A higher degree of DC differentiation led to a gradual increase of IL-12 secretion in the four groups. The difference was statistically significant (P<0.05) for this secretion (group D, 10,620.73±276.73 pg/ml). The expression levels of TRAF6 mRNA were significantly higher in group D than those in the other three groups (P<0.01). Although there was no significant difference in the expression levels of TRAF6 mRNA between groups B and C, the expression levels of TRAF6 mRNA between groups B and C were higher than those of the control group. The TRAF6 protein expression was higher in group D than that in the other three groups (P<0.01), and the difference was statistically significant. There was a statistically significant difference in the TRAF6 protein expression between group A and groups B and C, but the expression in group C was higher than that in group B (P<0.01). In conclusion, the expression of co-stimulatory molecules gradually increased in the DCs of different maturation states, and the expression of IL-12, TRAF6 mRNA, and TRAF6 protein positively correlated with the degree of DC maturation. TRAF6 is important in iDC polarity and maturation.

Effect of Dezocine on IL-12 and IL-10 secretion and lymphocyte activation by culturing dendritic cells from human umbilical cord blood

Dezocine has been generally utilized for pain therapy and auxiliary anesthesia. Although it has some advantages on the prevention of some anesthesia related complications, its effect on immune responses remains unclear. Our study investigated the effects of Dezocine on IL-10 and IL-12 secretion and lymphocytes activation by culturing dendritic cells (DCs), and revealed the underlying mechanism. Mononuclear cells were divided into negative control group (GN), positive control group (GP), experimental group (GD; GD5, D7, D9). DCs morphological structure was performed by microscope and its phenotypes were evaluated by flow cytometry. IL-12 and IL-10 levels were determined by ELISA and lymphocyte proliferation capacity was performed by MTT assay. Results showed that typical morphological characters of DCs were observed in GP and GD. The positive cell percentages of CD83, HLA-DR, CD80, CD86 and CD40 in GD were lower than those in GP, but higher than the GN group (P<0.01). IL-12 level in GD was higher than in GP, however, IL-10 was opposite (P<0.01). The optical density in GD was lower than in GP (P<0.05). There were no dose-dependent relationships correlated with DCs phenotypes, IL-12 and IL-10 secretion and lymphocytes activation (P>0.05). Our conclusion was that Dezocine might play a role in immunity by regulating IL-12 and IL-10 secretion, and affecting lymphocyte activity in process of DCs maturation. Our findings reveal an unexpected immuno-regulatory function of Dezocine in DCs and provide an important insight for investigating the effect of opioid drugs in immunologic responses.

Effects of Mycoplasma hyopneumoniae on porcine nasal cavity dendritic cells

Mycoplasma hyopneumoniae (Mhp) is the primary etiological agent responsible for swine enzootic pneumonia (EP), a disease that cause tremendous economic losses all over the swine industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium. DCs uptake and present antigens to T cells, to initiate protective immune responses or generate immune-mediated pathology in different infections. In this study, we investigated the changes in the different DCs subpopulations, T cells and SIgA positive cells counts in porcine nasal cavity after long time Mhp infection. We further evaluated the role of porcine DCs in Mhp exposure. Our results showed that the number of SLA-II-DR+SWC3a+DCs, SLA-II-DR+CD11b+ DCs, T cells, SIgA positive cells in nasal cavity were decreased after Mhp 28 days infection in vivo experiment. The antigen presenting ability of DCs were inhibited by Mhp exposure. DCs couldn’t activate T-cell proliferation by down-regulating the antigen presenting molecule CD1a expression and promoting high level of IL-10 production. Further more, the expression levels of IL-12 and IFN-γ in DCs were decreased, suggesting that DCs favour for Th2 immune response development after Mhp exposure in vitro. Taken together, Mhp infection impairs the immune function which allows the persistence of Mhp and cause predispose pigs to secondary infections. The decline of DCs presentation ability is the reason why dysfunction and persistence in Mhp infection. These findings are benefit for exploring the pathogenic mechanisms of Mhp in pigs.

Galectin-1 is essential for the induction of MOG35-55 -based intravenous tolerance in experimental autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE), intravenous (i.v.) injection of the antigen, myelin oligodendrocyte glycoprotein-derived peptide, MOG35-55 , suppresses disease development, a phenomenon called i.v. tolerance. Galectin-1, an endogenous glycan-binding protein, is upregulated during autoimmune neuroinflammation and plays immunoregulatory roles by inducing tolerogenic dendritic cells (DCs) and IL-10 producing regulatory type 1 T (Tr1) cells. To examine the role of galectin-1 in i.v. tolerance, we administered MOG35-55 -i.v. to wild-type (WT) and galectin-1 deficient (Lgals1(-/-) ) mice with ongoing EAE. MOG35-55 suppressed disease in the WT, but not in the Lgals1(-/-) mice. The numbers of Tr1 cells and Treg cells were increased in the CNS and periphery of tolerized WT mice. In contrast, Lgals1(-/-) MOG-i.v. mice had reduced numbers of Tr1 cells and Treg cells in the CNS and periphery, and reduced IL-27, IL-10, and TGF-β1 expression in DCs in the periphery. DCs derived from i.v.-tolerized WT mice suppressed disease when adoptively transferred into mice with ongoing EAE, whereas DCs from Lgals1(-/-) MOG-i.v. mice were not suppressive. These findings demonstrate that galectin-1 is required for i.v. tolerance induction, likely via induction of tolerogenic DCs leading to enhanced development of Tr1 cells, Treg cells, and downregulation of proinflammatory responses.

IL-10 promotes homeostatic proliferation of human CD8(+) memory Tcells and, when produced by CD1c(+) DCs, shapes naive CD8(+) T-cell priming.

IL-10 is an anti-inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8(+) CTL, IL-10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL-10 produced by human APC subsets in T-cell responses. IL-10 production was restricted to CD1c(+) DCs and CD14(+) monocytes. Interestingly, it was differentially regulated, since R848 induced IL-10 in DCs, but inhibited IL-10 in monocytes. Autocrine IL-10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high-affinity peptides. Nevertheless, it completely blocked cross-priming and priming with low-affinity peptides of a self/tumor-antigen. IL-10 also inhibited CD1c(+) DC-induced CD4(+) T-cell priming and enhanced Foxp3 induction, but was insufficient to induce T-cell IL-10 production. CD1c(+) DC-derived IL-10 had also no effect on DC-induced secondary expansions of memory CTL. However, IL-15-driven, TCR-independent proliferation of memory CTL was enhanced by IL-10. We conclude that DC-derived IL-10 selects high-affinity CTL upon priming. Moreover, IL-10 preserves established CTL memory by enhancing IL-15-dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL-10 promotes CTL responses in humans.

IRF5 as an upstream regulator of severe steroid-refractory asthma.

Abstract Severe, corticosteroid (CS)-refractory asthma is a significant problem both clinically and in terms of economic impact. A CS-responsive T helper-type 2 (Th2)/Type 2 response is a feature of milder asthma. However, we recently reported heightened airway type 1 (IFN-γ) and type 17 (IL-17) immune responses along with detectable Th2 response in &amp;gt;50% of clinically diagnosed severe asthma cases in adults in the context of CS treatment. In macrophages, the transcription factor IRF5 was previously shown to promote the expression of Type 1/17-inducing cytokines. However, its role in promoting the expression of these cytokines in dendritic cells (DCs), which would impact T helper cell differentiation, has not been studied. Thus, to determine the role of IRF5 in eliciting a CS-refractory severe asthma phenotype, we utilized our newly established model of severe asthma, in which IFN-γ plays an important role in increased airway hyperresponsiveness (AHR), and which is poorly responsive to CS mimicking human disease. Using WT and IRF5−/− mice subjected to the severe asthma model, we show that IRF5 is required to promote Th1 and Th17 immune responses and importantly, also plays a role in increased AHR. In the absence of IRF5, reduced Th1/Th17 responses were associated with decreased expression of the type-1/17-promoting cytokines IL-12 and IL-6 in lung DCs. Also, using bronchoalveolar lavage (BAL) cells, we detected higher IRF5 expression in human severe versus milder asthma. Therefore, IRF5 may represent a potential target for therapeutic intervention in CS-refractory human severe asthma with a strong Th1/Th17 immunobiologic component.

β2-adrenoceptor signaling reduction in dendritic cells is involved in the inflammatory response in adjuvant-induced arthritic rats.

Rheumatoid arthritis (RA) is characterized by inflammation of the synovium, which leads to the progressive destruction of cartilage and bone. Adrenoreceptor (AR) signaling may play an important role in modulating dendritic cell (DC), which may be involved in the pathogenesis of RA. We examined the effect of the β-AR agonist isoprenaline (ISO) on DC function, the impact of the β2-AR agonist salbutamol on adjuvant-induced arthritic (AA) rats, and changes in β2-AR signaling in DCs during the course of AA. ISO inhibited the expression of the surface molecules CD86 and MHC-II, inhibited the stimulation of T lymphocyte proliferation by DC and TNF-α secretion, and promoted DC antigen uptake and IL-10 secretion. The effects of ISO on MHC-II expression, DC stimulation of T lymphocyte proliferation, and DC antigen uptake were mediated by β2-AR. Treatment with salbutamol ameliorated the severity of AA and histopathology of the joints and inhibited proliferation of thymus lymphocytes and FLS in vivo. β2-AR signaling was weaker in AA rats compared to the control. Elevated GRK2 and decreased β2-AR expression in DC cytomembranes were observed in AA and may have decreased the anti-inflammatory effect of β2-AR signaling. Decreased β2-AR signaling may be relevant to the exacerbation of arthritis inflammation.

Abstract A157: Oncolytic poliovirus mediated immune events

Abstract Introduction: Tumor-targeted therapies that efficiently eliminate malignant cells and in the process engage the innate and adaptive immune system are desirable for preventing cancer recurrence. We have pioneered an oncolytic poliovirus therapy, PVSRIPO, which selectively targets and eliminates malignant cells. PVSRIPO is a recombinant polio:rhinovirus chimera, engineered to eliminate neuronal competence and is devoid of neuropathogenicity after intracerebral inoculation in non-human primates and in humans. PVSRIPO has cancer tropism due to widespread ectopic expression of the poliovirus receptor, CD155, in solid cancers. PVSRIPO kills tumor cells with no effect on normal cells. Unlike other oncolytic viruses, PVSRIPO is effective in the presence of neutralizing antibodies and an innate antiviral response. A first-in-human Phase-1 study with PVSRIPO at Duke University has shown remarkable promise in patients with recurrent glioblastoma (GBM), a uniformly lethal disease. PVSRIPO tumor cell killing is associated with the induction of danger- and pathogen-associated molecular patterns (DAMPs and PAMPs) via antiviral type I interferons (IFNs) and simultaneous non-lethal infection of antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs) via CD155. Type I IFNs are known to inhibit tumor proliferation, activate innate immune cells and bridge innate and adaptive immunity among other functions. Methods: To understand immune events associated with poliovirus infection of APCs, we examined the effects of PVSRIPO treatment on the human macrophage cell line (THP1) and primary human monocyte-derived DCs. PVSRIPO-treated DCs were evaluated for expression of maturation/activation markers and compared to untreated immature and mature DCs. To investigate if DCs exposed to PVSRIPO-induced dying tumor cells are capable of presenting tumor antigens to T cells, we performed an in vitro human immunotherapy assay. Human DCs generated from HLA-A2+ donor cells were incubated with PVSRIPO-induced tumor cell lysate and then used to stimulate autologous T cells in vitro followed by a cytotoxic T lymphocyte (CTL) assay. All tumor cell lines used for the human immunotherapy experiments were positive for HLA-A2 expression. The following tumor cell lines were used for this analysis: DM6 (MART+) melanoma cell line, MDA-MB231 (CEA+) triple-negative breast cancer cell line, SUM149 (EGFR+) inflammatory breast cancer cell line and LNCaP (PSA+) prostate cancer cell line. Results: PVSRIPO infection of THP1 macrophages induces Mda5/Stat1 and Stat1 phosphorylation as well as TNFα and IFNβ release. Coculturing of DCs with PVSRIPO-induced tumor cell lysate stimulates DC activation and IL12 production. Human DCs loaded with PVSRIPO-induced tumor cell lysate are capable of stimulating tumor antigen-specific cytotoxic T cells in vitro. Autologous DCs transfected with RNA that encodes for CEA, EGFR, MART or PSA (tumor antigen-expressing cells) and human tumor cell lines were used as targets to assess tumor antigen-specificity of T cells. Conclusion: Our data suggests that along with destruction of the primary tumor, oncolytic poliovirus mediates immune events. Importantly, we demonstrate that human DCs co-incubated with PVSRIPO-induced tumor cell lysate stimulate tumor antigen-specific cytotoxic T cells in an in vitro human immunotherapy assay. In ongoing studies we are analyzing oncolytic poliovirus mediated immune events in syngeneic, immunocompetent murine models of breast cancer and prostate cancer. Citation Format: Michael C. Brown, Eda K. Holl, David Boczkowski, Darell D. Bigner, Matthias Gromeier, Smita K. Nair. Oncolytic poliovirus mediated immune events. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A157.

Synthesized peptides 705–734 from hepatitis C virus E2 glycoprotein induce dendritic cell maturation by activating p38 MAPK signaling

Hepatitis C virus (HCV) envelope E2 is a glycoprotein that are implicated in HCV infection by facilitating its entry and immune evasion, which in turn leads to hepatitis, hepatocellular carcinoma and other chronic liver diseases. It is reported that the HCV E2 stem region comprise a functional region for HCV entry; however, the roles and underlying mechanism of these conserved residues on the E2 protein in the immune response after HCV infection are still not well defined. In this study, we synthesized 30 aa peptides containing residues 705–734 (E2-705) of HCV E2 using the solid-phase peptide synthesis (SPPS) method. The characteristics of the synthesized peptides were identified by Western blot and cell culture derived HCV particles (HCVcc) infection blocking assay. ELISA and flow cytometry assays were employed to determine the effect of the synthesized peptide on dendritic cells (DCs) response and CD4+ T cell activation. Results showed that the synthesized E2-705 peptides binds to DCs by interaction with DC-SIGN receptor. E2-705 peptides induced the maturation of infected DCs to a similar extent with recombinant HCV E2 as reflected by the antigen uptake potential and allostimulatory capacity. Furthermore, the E2-705 peptides increased the production of IL-12, CD80 and CD86 but reduced the IL-10 in DCs, in which p38 MAPK signaling might be involved. These results suggest that the carboxyl-terminus beyond the core ectodomain of HCV E2 protein may play a key role in immunoreaction of HCV infection, giving a new understanding of HCV E2 and a novel target for the design of HCV vaccines or inhibitors.

Suppression of Dendritic Cell-Derived IL-12 by Endogenous Glucocorticoids Is Protective in LPS-Induced Sepsis.

Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GRCD11c-cre) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GRCD11c-cre mice, CD8+ DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c+ cells rescued LPS-induced loss of CD8+ DCs but not other DC subsets. Unlike wild-type animals, exposure of GRCD11c-cre mice to low-dose LPS did not induce CD8+ DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.

Soluble Mediators in Platelet Concentrates Modulate Dendritic Cell Inflammatory Responses in an Experimental Model of Transfusion.

The transfusion of platelet concentrates (PCs) is widely used to treat thrombocytopenia and severe trauma. Ex vivo storage of PCs is associated with a storage lesion characterized by partial platelet activation and the release of soluble mediators, such as soluble CD40 ligand (sCD40L), RANTES, and interleukin (IL)-8. An in vitro whole blood culture transfusion model was employed to assess whether mediators present in PC supernatants (PC-SNs) modulated dendritic cell (DC)-specific inflammatory responses (intracellular staining) and the overall inflammatory response (cytometric bead array). Lipopolysaccharide (LPS) was included in parallel cultures to model the impact of PC-SNs on cell responses following toll-like receptor-mediated pathogen recognition. The impact of both the PC dose (10%, 25%) and ex vivo storage period was investigated [day 2 (D2), day 5 (D5), day 7 (D7)]. PC-SNs alone had minimal impact on DC-specific inflammatory responses and the overall inflammatory response. However, in the presence of LPS, exposure to PC-SNs resulted in a significant dose-associated suppression of the production of DC IL-12, IL-6, IL-1α, tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein (MIP)-1β and storage-associated suppression of the production of DC IL-10, TNF-α, and IL-8. For the overall inflammatory response, IL-6, TNF-α, MIP-1α, MIP-1β, and inflammatory protein (IP)-10 were significantly suppressed and IL-8, IL-10, and IL-1β significantly increased following exposure to PC-SNs in the presence of LPS. These data suggest that soluble mediators present in PCs significantly suppress DC function and modulate the overall inflammatory response, particularly in the presence of an infectious stimulus. Given the central role of DCs in the initiation and regulation of the immune response, these results suggest that modulation of the DC inflammatory profile is a probable mechanism contributing to transfusion-related complications.

Ginseng Berry Extract Promotes Maturation of Mouse Dendritic Cells.

Ginseng extract has been shown to possess certain anti-virus, anti-tumor and immune-activating effects. However, the immunostimulatory effect of ginseng berry extract (GB) has been less well characterized. In this study, we investigated the effect of GB on the activation of mouse dendritic cells (DCs) in vitro and in vivo. GB treatment induced up-regulation of co-stimulatory molecules in bone marrow-derived DCs (BMDCs). Interestingly, GB induced a higher degree of co-stimulatory molecule up-regulation than ginseng root extract (GR) at the same concentrations. Moreover, in vivo administration of GB promoted up-regulation of CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen DCs. GB also promoted the generation of Th1 and Tc1 cells. Furthermore, Toll like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling pathway were essential for DC activation induced by GB. In addition, GB strongly prompted the proliferation of ovalbumin (OVA)-specific CD4 and CD8 T cells. Finally, GB induced DC activation in tumor-bearing mice and the combination of OVA and GB treatment inhibited B16-OVA tumor cell growth in C57BL/6 mice. These results demonstrate that GB is a novel tumor therapeutic vaccine adjuvant by promoting DC and T cell activation.

Captopril inhibits maturation of dendritic cells and maintains their tolerogenic property in atherosclerotic rats

Atherosclerosis (AS) is a systemic disease of the immune system featuring hyperactive dendritic cells (DCs) in atherosclerotic plaques and organs. Captopril, a representative medicine of angiotensin-converting enzyme inhibitors, has been demonstrated to be effective in treating AS. However, captopril's anti-atherosclerotic mechanism is still poorly understood. Therefore, this study was primarily performed to investigate the effects of captopril on the function of DCs in vivo. AS in rats was induced by feeding them with atherogenic diets, and it was evaluated by the levels of plasma lipids and aortic cholesterol. DCs' activity was appraised by endocytic activity, mixed lymphocyte reactions and cytokine secretion. The markers of DCs (CD103, CD80, CD86 and MHC-II) and Treg (CD4+, CD25+ and Foxp3+) were assayed by western blotting analysis and flow cytometry. Cytokine level was measured by an enzyme-linked immunosorbent assay. The results showed that captopril treatment (10, 20mg/kg/d) obviously improved dyslipidemia and reduced the levels of aortic cholesterol. Captopril significantly reduced CD103, CD80, CD86 and MHC-II protein expression while increasing that of Foxp3 in aortic tissue. Further study indicated oral administration of captopril up-regulated endocytic activity and reduced the immunostimulatory function of splenic DCs. Captopril treatment also promoted IL-10 & TGF-β production while decreasing that of IL-6 & IL-12 in splenic DCs. Finally, the results of flow cytometry indicated that captopril obviously inhibited DC maturation and promoted Treg polarization. Captopril treatment was able to inhibit DC maturation and maintain their tolerogenic property, which is closely associated with DC anti-atherosclerosis activity.

MTOR regulates TLR-induced c-fos and Th1 responses to HBV and HCV vaccines.

Although IL-12 plays a critical role in priming Th1 and cytotoxic T lymphocyte (CTL) responses, Toll-like receptor (TLR) signaling only induces low amounts of IL-12 in dendritic cells and macrophages, implying the existence of stringent regulatory mechanisms. In this study, we sought to uncover the mechanisms underlying TLR-induced IL-12 expression and the Th1 response. By systemic screening, we identified a number of protein kinases involved in the regulation of TLRinduced IL-12 expression. In particular, PI3K, ERK, and mTOR play critical roles in the TLR-induced Th1 response by regulating IL-12 and IL-10 production in innate immune cells. Moreover, we identified c-fos as a key molecule that mediates mTOR-regulated IL-12 and IL-10 expression in TLR signaling. Mechanistically, mTOR plays a crucial role in c-fos expression, thereby modulating NFκB binding to promoters of IL-12 and IL-10. By controlling the expression of a special innate gene program, mTOR can specifically regulate the TLR-induced T cell response in vivo. Furthermore, blockade of mTOR by rapamycin efficiently boosted TLR-induced antigen-specific T and B cell responses to HBV and HCV vaccines. Taken together, these results reveal a novel mechanism through which mTOR regulates TLR-induced IL-12 and IL-10 production, contributing new insights for strategies to improve vaccine efficacy.

Interleukin-15 Dendritic Cells Harness NK Cell Cytotoxic Effector Function in a Contact- and IL-15-Dependent Manner.

The contribution of natural killer (NK) cells to the treatment efficacy of dendritic cell (DC)-based cancer vaccines is being increasingly recognized. Much current efforts to optimize this form of immunotherapy are therefore geared towards harnessing the NK cell-stimulatory ability of DCs. In this study, we investigated whether generation of human monocyte-derived DCs with interleukin (IL)-15 followed by activation with a Toll-like receptor stimulus endows these DCs, commonly referred to as “IL-15 DCs”, with the capacity to stimulate NK cells. In a head-to-head comparison with “IL-4 DCs” used routinely for clinical studies, IL-15 DCs were found to induce a more activated, cytotoxic effector phenotype in NK cells, in particular in the CD56bright NK cell subset. With the exception of GM-CSF, no significant enhancement of cytokine/chemokine secretion was observed following co-culture of NK cells with IL-15 DCs. IL-15 DCs, but not IL-4 DCs, promoted NK cell tumoricidal activity towards both NK-sensitive and NK-resistant targets. This effect was found to require cell-to-cell contact and to be mediated by DC surface-bound IL-15. This study shows that DCs can express a membrane-bound form of IL-15 through which they enhance NK cell cytotoxic function. The observed lack of membrane-bound IL-15 on “gold-standard” IL-4 DCs and their consequent inability to effectively promote NK cell cytotoxicity may have important implications for the future design of DC-based cancer vaccine studies.

Multi-enzyme aminoacyl tRNA synthetase complex component AIMp1/p43 as an important Th1 mediator (IRC7P.428)

Abstract Of professional antigen presenting cells, only the dendritic cells (DC) are regarded as initiators of adaptive immune responses. Previously we identified a Th1 polarizing phenotype from DCs loaded with overlapping (homologous) MHC class I and II determinants, including augmented DC IL-12 production, generation of CD8+ cytolytic effectors, and T cell IFNγ secretion, suggesting DC intrinsic mechanisms to recognize and compare antigenic epitopes. AIMp1/p43 is a structural component of the multi-enzyme aminoacyl-tRNA synthetase complex (mARS) and indicated by previous studies to be a pro-inflammatory cytokine. We identified high levels of p43 release correlated with degree of homology between antigenic epitopes on DC class I and II molecules. In vitro studies confirmed p43 deficiency impairs DC costimulatory and Th1 polarizing functions. It also reversely regulates the production of IFNγ and IL4 within T cells. In vivo, p43 deficiency abolishes the ability of DC to mediate effective response against B16 melanoma tumors, and leads to higher susceptibility to influenza viral infections. These data support our hypothesis that p43 is a critical Th1 polarizing effector molecule. We will investigate further into p43 functions in DC and T cell crosstalk, targeting candidate cell surface receptors and intracellular signaling pathways. Additionally, we will better characterize in vivo roles of p43 in anti-tumor and anti-viral models and seek to apply it as a novel vaccination adjuvant.

Low IL-10/TNFα ratio in patients with coronary artery disease and reduced left ventricular ejection fraction with a poor prognosis after 10 years.

Monocytes and dendritic cells (DC) produce tumour necrosis factor (TNF)α during inflammatory processes, but secrete interleukin (IL)-10 simultaneously in order to balance the pro-inflammation. In the present study, we investigated the expression of TNFα and IL-10 by monocytes and DC in patients with a poor cardiovascular prognosis after 10 years. Peripheral blood monocytes were isolated from 30 patients with coronary artery disease (CAD) with stable angina pectoris (SAP), or with an acute coronary syndrome (ACS). Monocytes were differentiated over 7 days to DC. Intracellular accumulation of TNFα and IL-10 in monocytes and DC was analysed by flow cytometry and correlated with the heart function, total and cardiovascular (CV) mortality, as well as with cardiovascular event rate over 10 years. We observed a decreased left ventricular function (LV-EF) for both SAP and ACS patients (p<0.01), as well as a reduced IL-10/TNFα ratio for monocytes (p=0.01) and DC (p<0.01) for both patient groups in comparison to age-matched control group. Only the IL-10/TNFα ratio for monocytes correlated with LV-EF (r=0.4302; p<0.01). Patients with a low LV-EF as well as patients with a low IL-10/TNFα ratio showed an increased cardiovascular mortality over 10 years (both p<0.05). The IL-10/TNFα ratio is decreased in patients with low ejection fraction and poor prognosis. The reduced heart function correlates with an increased proinflammatory state (low monocytic IL-10/TNFα ratio) in patients with CAD. This observed imbalance of IL-10 and TNFα in monocytes might explain pathophysiological processes in atherosclerosis and heart failure.

P100, a precursor of NF-κB2, inhibits c-Rel and reduces the expression of IL-23 in dendritic cells

Nuclear factor κB regulates various genes involved in the immune response, inflammation, cell survival, and development. NF-κB activation is controlled by proteins possessing ankyrin repeats, such as IκBs. A precursor of the NF-κB2 (p52) subunit, p100, contains ankyrin repeats in its C-terminal portion and has been found to act as a cytoplasmic inhibitor of RelA in the canonical pathway of NF-κB activation. Here, we demonstrate that p100 also suppresses c-Rel function in dendritic cells. Expression of the p19 and p40 subunits of IL-23, a c-Rel-dependent cytokine, was enhanced in p100-deficient cells, although expression of a RelA-dependent cytokine, TNF-α, was reduced. Nuclear translocation of c-Rel was enhanced in p100-deficient cells. p100, and not the processed p52 form, associated with c-Rel in the steady state and dissociated immediately after lipopolysaccharide stimulation in wild-type dendritic cells. Four hours after the stimulation, p100 was newly synthesized and associated with c-Rel again. In cells expressing both c-Rel and RelA, c-Rel is preferentially suppressed by p100.