Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.     

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+) T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.     

STAT3 Knockdown in B16 Melanoma by siRNA Lipopolyplexes Induces Bystander Immune Response In Vitro and In Vivo

Persistent activation of STAT3 plays a major role in cancer progression and immune escape. Therefore, targeting STAT3 in tumors is essential to enhance/reactivate antitumor immune response. In our previous studies, we demonstrated the efficacy of stearic acid-modified polyethylenimine (PEI-StA) in promoting small interfering RNA (siRNA) silencing of STAT3 in B16.F10 melanoma in vitro and in vivo. In the current study, we examine the immunologic impact of this intervention. Toward this goal, the infiltration and activation of lymphocytes and dendritic cells (DCs) in the tumor mass were assessed using flow cytometry. Moreover, the levels of IFN-γ, IL-12, and TNF-α in homogenized tumor supernatants were determined. Moreover, mixed lymphocytes reaction using splenocytes of tumor-bearing mice was used to assess DC functionality on siRNA/lipopolyplexes intervention. Our results demonstrated up to an approximately fivefold induction in the infiltration of CD3(+) cells in tumor mass on STAT3 knockdown with high levels of CD4(+), CD8(+), and NKT cells. Consistently, DC infiltration in tumor milieu increased up to approximately fourfold. Those DCs were activated, in an otherwise suppressive microenvironment, as evidenced by a high expression of costimulatory molecules CD86 and CD40. ELISA analysis revealed a significant increase in IFN-γ, IL-12, and TNF-α. Moreover, mixed lymphocytes reaction demonstrated alloreactivity of these DCs as assessed by high T-cell proliferation and IL-2 production. Our results suggest a bystander immune response after local STAT3 silencing by siRNA. This strategy could be beneficial as an adjuvant therapy along with current cancer vaccine formulations.     

Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

IFN-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes is promoted by NK cells via MIP-1β and LFA-1

Several systemic autoimmune diseases display a prominent IFN signature. This is caused by a continuous IFN-α production by plasmacytoid dendritic cells (pDCs), which are activated by immune complexes (ICs) containing nucleic acid. The IFN-α production by pDCs stimulated with RNA-containing IC (RNA-IC) consisting of anti-RNP autoantibodies and U1 small nuclear ribonucleoprotein particles was recently shown to be inhibited by monocytes, but enhanced by NK cells. The inhibitory effect of monocytes was mediated by TNF-α, PGE(2), and reactive oxygen species, but the mechanisms for the NK cell-mediated increase in IFN-α production remained unclear. In this study, we investigated the mechanisms whereby NK cells increase the RNA-IC-induced IFN-α production by pDCs. Furthermore, NK cells from patients with systemic lupus erythematosus (SLE) were evaluated for their capacity to promote IFN-α production. We found that CD56(dim) NK cells could increase IFN-α production >1000-fold after RNA-IC activation, whereas CD56(bright) NK cells required costimulation by IL-12 and IL-18 to promote IFN-α production. NK cells produced MIP-1α, MIP-1β, RANTES, IFN-γ, and TNF-α via RNA-IC-mediated FcγRIIIA activation. The IFN-α production in pDCs was promoted by NK cells via MIP-1β secretion and LFA-mediated cell-cell contact. Moreover, NK cells from SLE patients displayed a reduced capacity to promote the RNA-IC-induced IFN-α production, which could be restored by exogenous IL-12 and IL-18. Thus, different molecular mechanisms can mediate the NK cell-dependent increase in IFN-α production by RNA-IC-stimulated pDCs, and our study suggests that the possibility to therapeutically target the NK-pDC axis in IFN-α-driven autoimmune diseases such as SLE should be investigated.     

Down regulation of CD24 and HER-2/neu in breast carcinoma cells by activated human dendritic cell. Role of STAT3

Human dendritic cells (DCs) stimulated with cytokines and LPS down regulate the expression of proto-oncogene HER-2/neu and GPI linked protein CD24 in breast cancer cell lines. We demonstrated that na?ve DC from human peripheral blood, when stimulated with IFN-γ, IL-15 or LPS reduces the expression of HER-2/neu and CD24, via activation of TNF-α. Pretreatment of tumor cells with STAT3 specific inhibitors or knocking down of STAT3 by SiRNA makes the tumor cell more susceptible to apoptosis and DC mediated inhibition of both CD24 and HER-2/neu. Thus DC could acts as an inhibitory regulator in suppressing oncogene and prevention of metastasis.     

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-β-d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.     

Cell type specificity and host genetic polymorphisms influence antibody-dependent enhancement of dengue virus infection

Antibody-dependent enhancement (ADE) is implicated in severe, usually secondary, dengue virus (DV) infections. Preexisting heterotypic antibodies, via their Fc-gamma receptor (FcγR) interactions, may increase disease severity through enhanced target cell infection. Greater numbers of infected target cells may contribute to higher viremia and excess cytokine levels often observed in severe disease. Monocytes, macrophages, and immature and mature dendritic cells (DC) are considered major cellular targets of DV. Apheresis of multiple donors allowed isolation of autologous primary myeloid target cell types for head-to-head comparison of infection rates, viral output, and cytokine production under direct infection (without antibody) or ADE conditions (with antibody). All studied cell types except immature DC supported ADE. All cells undergoing ADE secreted proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) at enhancement titers, but distinct cell-type-specific patterns were observed for other relevant proteins (alpha/beta interferon [IFN-α/β] and IL-10). Macrophages produced type I interferons (IFN-α/β) that were modulated by ADE. Mature DC mainly secreted IFN-β. Interestingly, only monocytes secreted IL-10, and only upon antibody-enhanced infection. While ADE infection rates were remarkably consistent in monocytes (10 to 15%) across donors, IL-10 protein levels varied according to previously described regulatory single nucleotide polymorphisms (SNPs) in the IL-10 promoter region. The homozygous GCC haplotype was associated with high-level IL-10 secretion, while the ACC and ATA haplotypes produced intermediate and low levels of IL-10, respectively. Our data suggest that ADE effects are cell type specific, are influenced by host genetics, and, depending on relative infection rates, may further contribute to the complexity of DV pathogenesis.     

Effects of the Linear Fragments of Beta-(1→3)-Glucans on Cytokine Production <Emphasis Type="Italic">in vitro</Emphasis>

Beta-glucans, homopolysaccharides composed of 3,6-branching β-(1→3)-D-glucan chains, attract great interest as inducers of cytokine synthesis. In this work, we studied the ability of linear fragments of beta-glucan chains to activate cytokine synthesis. Synthetic nona-β-(1→3)-D-glucoside (SO) representing a linear fragment of beta-glucan chain, endotoxin (ED), and natural β-(1→3)-D-glucan (GL) were tested for their role as inducers of cytokines in whole peripheral blood cultures collected from 17 individuals. The concentrations of IL-12p70, IFN-γ, IL-2, IL-10, IL-8, IL-6, IL-4, IL-5, IL-1β, TNF-α, and TNF-β were measured in the supernatants after 2, 24, and 48 h of cell culturing. SO, ED, and GL stim- ulated production of pro-inflammatory IFN-γ, IL-1β, IL-2, IL-6, IL-8, TNF-α and anti-inflammatory IL-10. The high- est levels of biosynthesis after stimulation with SO were registered for IL-6, IL-8, and TNF-α. SO stimulated production of all cytokines (except IFN-γ) to a lesser extent than ED and GL. The IFN-γ/IL-10 (Th1/Th2) ratios after 24 and 48 h of culturing were 3.1 and 7.5 for SO; 0.03 and 0.1 for GL; and 0.06 and 0.2 for ED, respectively. The results indicate that lin- ear fragments of beta-glucans cause a more pronounced shift of immune response towards the pro-inflammatory (Th1) type than beta-glucan itself.     

Escherichia coli heat-labile enterotoxin promotes protective Th17 responses against infection by driving innate IL-1 and IL-23 production

Escherichia coli heat-labile enterotoxin (LT) is a powerful mucosal adjuvant; however, it is associated with toxic effects when delivered intranasally, and its mechanism of action is poorly understood. In this article, we demonstrate that LT acts as a highly effective adjuvant when administered parenterally, promoting Ag-specific IL-17, as well as IFN-γ, IL-4, and IL-10 production in response to coadministered Ags. We found that the adjuvant activity of LT was mediated in part by inducing dendritic cell (DC) activation; LT promoted CD80 and CD86 expression by DCs and enhanced IL-1α, IL-1β, and IL-23 production. An LT mutant, LTK63, that lacks enzyme activity was less effective than the wild-type toxin in promoting DC maturation and the development of Ag-specific Th17 cells. LT enhanced IL-23 and IL-1α production from DCs via activation of ERK MAPK and IL-1β secretion through activation of caspase-1 and the NLRP3 inflammasome. These cytokines played a major role in promoting Th17 responses by LT and LTK63. The induction of Th17 cells in vivo in response to LT and LTK63 as adjuvants was significantly reduced in IL-1RI-deficient mice. Finally, using a murine respiratory infection model, we demonstrated that LT can act as a highly effective adjuvant for a pertussis vaccine, promoting Ag-specific Th17 cells and protection against Bordetella pertussis challenge, which was significantly reduced in IL-17-defective mice. Our findings provide clear evidence that LT can promote protective immune responses in part through induction of innate IL-1 and, consequently, Th17 cells.     

Immunocompetent cell functions in Ph+ acute lymphoblastic leukemia patients on prolonged Imatinib maintenance treatment

Imatinib mesylate (Imatinib) is a potent inhibitor of defined tyrosine kinases and is effectively used for the treatment of malignancies characterized by the constitutive activation of these tyrosine kinases, such as Philadelphia chromosome-positive (Ph(+)) leukemias and gastrointestinal stromal tumors. Suppressive as well as stimulating effects of this drug on T lymphocytes or dendritic cells (DC), which play a major role in immune tumor surveillance, have been reported. For this reason, we questioned whether Imatinib could also affect the phenotypic and functional properties of these subpopulations in Ph(+) acute lymphoblastic leukemia (ALL) patients on prolonged Imatinib maintenance treatment. Circulating T lymphocytes and NK cells from Imatinib-treated Ph(+) ALL patients showed a subset distribution comparable to that of healthy donors. In addition, T-cell immunomodulant cytokine production (IFN-γ, TNF-α) and proliferative responses were not impaired. A normal monocyte-derived DC differentiation and apoptotic body loading capacity was also observed in the majority of Imatinib-treated patients. In contrast, an impairment in the DC intracellular production of IL-12 was recorded, although this was not observed when normal DC were exposed in vitro to Imatinib. Finally, in vivo Imatinib treatment did not affect the T-lymphocyte proliferation and IFN-γ production induced by leukemic apoptotic body-loaded DC, underling the potential capability of these cells to generate a specific immune response against tumoral antigens. Taken together, these findings provide evidence that immunotherapeutic approaches aimed at controlling residual disease in Ph(+) ALL patients in hematologic remission are not jeopardized by the long-term administration of Imatinib.     

TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection

Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activate DC and the expression patterns of TLR3 protein in DC subsets have not been established. In this article, we show that cross-priming CD8α(+) and CD103(+) DC subsets express much greater levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1(+) endosomes on stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of ～90 bp was sufficient to induce CD86, IL-12p40, IFN-β, TNF-α, and IL-6 expression, and to mature DC into APC that cross-presented exogenous Ags to CD8(+) T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of Ag-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.     

New polyprenyl phosphate based preparation Fortepren® as promising cytokine regulationg antiviral remedy

Fortepren^®, a product of the phosphorylation of polyprenols from fir needles (with sodium polyprenyl phosphate being the main active ingredient), belongs to the class of antiviral drugs with immunomodulating activity. Fortepren^® may be used as the drug of choice in the treatment of herpes diseases. It was shown that treatment with Fortepren^® of patients with a chronic recurrent herpes infection after acute phase termination with acyclovir decreased the recurrence rate, as well as the severity of local symptoms. Fortepren^® treatment of patients with a high incidence of recurrent herpes infection led to an increase in the interferon-producing ability of leucocytes stimulated with NDV, as well as in the production of key cytokines (IL-1β, IL-15, MIP-1α, IFN-γ, IL-12 (p40), TNF-α, IFN-α2, IL-12 (p70), IL-6) taking part in the protection against viral infection. Data suggest that the action of the drug is directed, first of all, to the cells responsible for the natural resistance of the organism (macrophages, dendritic cells, etc.). The activation of natural immunity appears to be a leading mechanism of protection from herpesviral infection under the influence of polyprenyl phosphate.     

An accelerated,clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA–loaded dendritic cells for cancer vaccination

Background

Many efforts have been devoted to improve the performance of dendritic cell (DC)–based cancer vaccines. Ideally, a DC vaccine should induce robust type 1–polarized T-cell responses and efficiently expand antigen (Ag)-specific cytotoxic T-cells, while being applicable regardless of patient human leukocyte antigen (HLA) type. Production time should be short, while maximally being good manufacturing practice (GMP)–compliant. We developed a method that caters to all of these demands and demonstrated the superiority of the resulting product compared with DCs generated using a well-established “classical” protocol.

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.     

CD8＋CD122＋T细胞在脑缺血过程中作用的研究

目的：探讨CD8＋CD122＋T细胞在脑缺血过程中的作用及其机制。方法：线栓法建立小鼠大脑中动脉栓塞模型（MCAO）;激光共聚焦显微镜检测小鼠脑缺血组织中CD8＋CD122＋T细胞浸润情况;流式细胞仪检测脑缺血组织中CD8＋CD122＋T细胞／CD3＋T细胞的比例及脾和胸腺中CD8＋CD12TT细胞数量变化;RT—PCR方法检测CD8＋CD122＋T细胞对氧糖剥夺（Oxygen—glucosedeprivation,OGD）条件下星形胶质细胞表达TNF-α,IL-1β,IFN-γ的影响。结果：各时间点脑缺血组织中均有CD8＋CD122＋T胞浸润,且随脑缺血时间延长,缺血侧脑组织中CD8＋CD122＋T细胞／CD3＋T细胞比例逐渐增加,5d和7d组差异显著,与非缺血侧相比,P5d〈0．05,P7d〈0．05;MCAO小鼠脾及胸腺中CD8＋CD122＋T细胞呈现先增高后降低的趋势。星形胶质细胞经OGD处理后,与对照组相比,IFN-γ、TNF-α、IL—1β表达显著增高,PIFN-γ〈0．01、PTNF-α〈0．001、PIL-1β〈0．01;CD122-blocked组与CD8＋组相比,IFN-γ、TNF-α、IL-1β表达明显增高,PIFN-γ〈0．05、PINF-α〈0．05、PIL-1β〈0．01;CD8＋组与HBSS组相比,IFN-γ表达降低,P〈0．05;IL-1β表达有降低的趋势。结论：CD8＋CD122可细胞在脑缺血过程中发挥保护性作用,其保护作用通过CD122抑制星形胶质细胞TNF-α,IL-1β,IFN-γ炎症因子表达实现的。