Generation of tolerogenic dendritic cells using Lactobacillus rhamnosus and Lactobacillus delbrueckii as tolerogenic probiotics

Systemic lupus erythematosus (SLE) concurs with excessive uncontrolled inflammatory immune responses that lead to the loss of immune tolerance. Dendritic cells (DCs) are important and determinant immune cells that regulate immune responses. Tolerogenic DCs with regulatory markers and cytokines could induce regulatory immune cells and responses. Tolerogenic probiotics are capable of producing regulatory DCs from monocytes in in vitro conditions. The purpose of this study was to evaluate the effect of Lactobacillus delbrueckii and Lactobacillus rhamnosus on the production of DCs in an in vitro condition. Peripheral blood mononuclear cells were isolated from the healthy and SLE donors. Monocytes were cultured with optimized concentrations of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin 4 (IL‐4) to produce immature DCs (IDCs). An IDC uptake assay was performed, and IDCs of healthy and SLE donors were divided into three subgroups following 48 hours of treatment with GM‐CSF and IL‐4, along with L. delbrueckii, L. rhamnosus, and mixed probiotics for the production of tolerogenic DCs. The surface expression of Human Leukocyte Antigen‐antigen D Related (HLA‐DR), CD86, CD80, CD83, CD1a, and CD14 was analyzed using flow cytometry, and the gene expression levels of indoleamine 2,3‐dioxygenase (IDO), IL‐10, and IL‐12 were measured using real‐time polymerase chain reaction. We observed significantly reduced expression of costimulatory molecules and other surface markers in the probiotic‐induced mature DCs (MDCs) in both healthy and SLE donor groups in comparison with lipopolysaccharide (LPS)‐induced MDCs. In addition, the expression of IDO and IL‐10 increased, whereas IL‐12 decreased significantly in probiotic‐induced MDCs compared with LPS‐induced MDCs. IDCs and especially mature tolerogenic DC of SLE patients highly expressed IDO. The results of the current study suggested that live probiotics could modify properties of DCs to modulatory cells, which might contribute to the induction of tolerance and renovation of immune hemostasis.     

EDIII‐DENV3 nanospheres drive immature dendritic cells into a mature phenotype in an in vitro model

Domain III of E protein of dengue virus (DENV) is a target for vaccine development. Unfortunately, this protein based platform has low general immunogenicity. To circumvent this problem, the use of an adjuvant‐nanoparticle delivery system to facilitate immunogenicity of soluble DENV‐EDIII protein was investigated. One of the key features of this delivery system is its ability to simultaneously deliver antigens and exert adjuvanticity on specialized immune cells. In this study, N‐trimethyl chitosan (TMC) nanoparticles (NPs) were generated to be used as adjuvant and carrier for soluble E‐domain III of dengue virus serotype 3 (sEDIII‐D3). Using ionotropic gelation, purified sEDIII‐D3 was encapsulated into TMC NPs to form EDIII‐D3 TMC NPs. After optimization, EDIII‐D3 TMC particles exhibited a loading efficiency of 81% and a loading capacity of 41%. The immunogenicity of EDIII‐D3 TMC NPs was tested using monocyte‐derived dendritic cells (MoDCs). It was found that EDIII‐D3 TMC NPs were well taken up by MoDCs. In addition, EDIII‐D3 TMC NP treated MoDCs significantly upregulated maturation markers (CD80, CD83, CD86 and HLA‐DR) and induced secretion of various cytokines and chemokines (IFN‐α, IL‐1β, IL‐6, IL‐2, IL‐12p70, IFN‐γ, IL‐4, IL‐10, IL‐8, MCP‐1, macrophage inflammatory protein‐1β, granulocyte‐colony stimulating factor, granulocyte–macrophage colony‐stimulating factor and IL‐7). These results indicate that EDIII‐D3 TMC NPs are potent immunogens, at least in vitro , with the ability to induce maturation of DCs and highlight the potential use of TMC NPs for enhancing immunogenicity of a non‐replicating dengue vaccine.

     

Nerve growth factor promotes TLR4 signaling-induced maturation of human dendritic cells in vitro through inducible p75NTR 1

Nerve growth factor (NGF) has been shown to play important roles in the differentiation, function, and survival of immune cells, contributing to immune responses and pathogenesis of autoimmune diseases. Dendritic cells (DCs) are a potent initiator for immune and inflammatory responses upon recognition of pathogens via Toll-like receptors (TLR). However, expression of NGF and its receptors on human monocyte-derived DCs (MoDCs) and the role of NGF in the response of DCs to TLR ligands remain to be investigated. In the present study, we demonstrate that there were weak expressions of NGF and no expression of NGF receptors p140(TrkA) and p75(NTR) on human immature MoDCs, however, the expression of NGF and p75(NTR) on MoDCs could be significantly up-regulated by LPS in a dose- and time-dependent manner. NGF could markedly promote LPS-induced expression of HLA-DR, CD40, CD80, CD83, CD86, CCR7, secretion of IL-12p40 and proinflammatory cytokines IL-1, IL-6, TNF-alpha, and the T cell-stimulating capacity of MoDCs, indicating that NGF can promote LPS-induced DC maturation. The promoting effect of NGF on LPS-induced MoDCs maturation could be completely abolished by pretreatment of MoDCs with p75(NTR) antagonist, suggesting that LPS-induced p75(NTR) mediates the effect. Furthermore, increased activation of the p38MAPK and NF-kappaB pathways has been shown to be responsible for the NGF-promoted DC maturation. Therefore, NGF facilitates TLR4 signaling-induced maturation of human DCs through LPS-up-regulated p75(NTR) via activation of p38 MAPK and NF-kappaB pathways, providing another mechanism for the involvement of NGF in the immune responses and pathogenesis of autoimmune diseases.     

microRNA-181a represses ox-LDL-stimulated inflammatory response in dendritic cell by targeting c-Fos

Oxidized LDL (ox-LDL) activates dendritic cells (DCs), thereby initiating inflammation responses in atherosclerosis, yet the modulatory mechanisms remain unclear. MicroRNAs (miRNAs) are important regulators for DC functions. This study evaluated the regulation by miRNAs of the ox-LDL-induced DC immune response. In CD11c⁺ DCs from ApoE-deficient mice with hyperlipidemia, microRNA miR-181a was significantly up-regulated. In cultured bone marrow-derived DCs (BMDCs), ox-LDL promoted DC maturation and up-regulated miR-181a expression. Abundance of miR-181a attenuated ox-LDL-induced CD83 and CD40 expression, inhibited the secretion of interleukin (IL)-6 and TNF-α, and up-regulated IL-10, an important anti-inflammatory cytokine that was inhibited by ox-LDL. Inhibition of the endogenous miR-181a reversed the effects on CD83 and CD40 as well as the effects on IL-6 and TNF-α. The putative target genes of miR-181a were evaluated by gene ontology assessment, and the c-Fos-mediated inflammation pathway was identified. miR-181a targeted the 3′ untranslated region of c-Fos mRNA by luciferase experiments. Thus, abundance of miR-181a reduced c-Fos protein, whereas inhibition of miR-181a increased c-Fos protein in BMDCs. We therefore suggest that miR-181a attenuates ox-LDL-stimulated immune inflammation responses by targeting c-Fos in DCs.     

Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.     

Engagement of Fc epsilon RI on human monocytes induces the production of IL-10 and prevents their differentiation in dendritic cells.

The local cytokine environment and the presence of stimulatory signals determine whether circulating monocytes will finally acquire characteristics of dendritic cells (DCs) or macrophages. Because FcepsilonRI expressed on professional APCs, e.g., monocytes and DCs, has been suggested to play a key role in the pathophysiology of atopic diseases, we evaluated the effect of receptor ligation on the generation of monocyte-derived DCs (MoDCs). Aggregation of FcepsilonRI at the initiation of the IL-4-GM-CSF-driven differentiation resulted in the emergence of macrophage-like cells with a strong expression of the mannose receptor and a low level of CD1a and the DC-specific markers CD83 and the actin-bundling protein (p55). These cells sustained the ability to take up FITC-labeled Escherichia coli by phagocytosis and were significantly less efficient in stimulating purified allogeneic T cells. In addition, receptor ligation of FcepsilonRI at the beginning of the culture prevented the generation of MoDCs, mainly due to a dramatic increase in the IL-10 production. These results suggest that FcepsilonRI aggregation prevents the generation of CD1a(+) MoDCs and imply a novel pivotal function of this receptor in modulating the differentiation of monocytes.     

Mesothelioma Tumor Cells Modulate Dendritic Cell Lipid Content,Phenotype and Function

Dendritic cells (DCs) play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs) were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay), upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4⁺CD8α^- DCs, CD4^-CD8α^- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8⁺ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.     

Subpopulations of human dendritic cells display a distinct phenotype and bind differentially to proteins of the extracellular matrix

CD1a(pos) dendritic cells (DCs) and Langerhans cells (LCs) are highly specialized antigen-presenting cells mainly localized in the skin. Various cells have been identified as precursors of cutaneous DCs, but the definitive precursor subpopulations remain to be defined and characterized in detail. In this study, DCs were generated in vitro from monocytes (monocyte-derived DCs, MoDCs) and from CD34(pos) stem cells (CD34(pos) cell-derived DCs, CD34DCs). By virtue of their CD14 and CD1a expression, four CD34DC subpopulations were characterized while MoDCs contain three different subpopulations. Of these, CD14-expressing cells are considered to be precursors of fully differentiated DCs, which themselves are CD14(neg)CD1a(pos). Both, MoDCs and CD34DCs expressed the alpha integrins LFA-1, Mac-1, CR4, VLA-4, VLA-5 and the beta2 integrin CD18. CD34DCs and MoDCs were negative for VLA-3, whereas MoDCs, but not CD34DCs expressed VLA-6. Phenotypic and functional characterization of the cells generated herein at earlier time points revealed that DCs at day 3 of culture may reflect the in vivo situation more closely than at day 7. Adhesion of DC precursors to endothelial cells and to components of the extracellular matrix is a prerequisite for their migration towards the epidermis. To this end, we investigated adhesion of CD34DCs and MoDCs to components of the cutaneous extracellular matrix. Distinct DC subsets showed a differential binding pattern to proteins of the extracellular matrix. MoDCs and CD34DCs bound preferentially to laminin 332 via CD49f and to fibronectin via CD49e, but only weakly to laminin 111 or to collagens. While CD14(pos) cells preferentially bound to laminin 332, CD1a(pos) cells adhered to fibronectin. In summary, subpopulations of CD34DCs and MoDCs are phenotypically related to each other, but not identical and display differential binding to components of the extracellular matrix.     

Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

Excessive immune‐mediated inflammatory reaction plays a deleterious role in ventricular remodelling after myocardial infarction (MI). Interleukin (IL)‐38 is a newly characterized cytokine of the IL‐1 family and has been reported to exert a protective effect in some autoimmune diseases. However, its role in cardiac remodelling post‐MI remains unknown. In this study, we found that the expression of IL‐38 was increased in infarcted heart after MI induced in C57BL/6 mice by permanent ligation of the left anterior descending artery. In addition, our data showed that ventricular remodelling after MI was significantly ameliorated after recombinant IL‐38 injection in mice. This amelioration was demonstrated by better cardiac function, restricted inflammatory response, attenuated myocardial injury and decreased myocardial fibrosis. Our results in vitro revealed that IL‐38 affects the phenotype of dendritic cells (DCs) and IL‐38 plus troponin I (TNI)‐treated tolerogenic DCs dampened adaptive immune response when co‐cultured with CD4⁺T cells. In conclusion, IL‐38 plays a protective effect in ventricular remodelling post‐MI, one possibility by influencing DCs to attenuate inflammatory response. Therefore, targeting IL‐38 may hold a new therapeutic potential in treating MI.     

Thymus‐derived regulatory T cells restrain pro‐inflammatory Th1 responses by downregulating CD70 on dendritic cells

The severity and intensity of autoimmune disease in immune dysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX) patients and in scurfy mice emphasize the critical role played by thymus‐derived regulatory T cells (tTregs) in maintaining peripheral immune tolerance. However, although tTregs are critical to prevent lethal autoimmunity and excessive inflammatory responses, their suppressive mechanism remains elusive. Here, we demonstrate that tTregs selectively inhibit CD27/CD70‐dependent Th1 priming, while leaving the IL‐12‐dependent pathway unaffected. Immunized mice depleted of tTregs showed an increased response of IFN‐γ‐secreting CD4⁺ T cells that was strictly reliant on a functional CD27/CD70 pathway. In vitro studies revealed that tTregs downregulate CD70 from the plasma membrane of dendritic cells (DCs) in a CD27‐dependent manner. CD70 downregulation required contact between Tregs and DCs and resulted in endocytosis of CD27 and CD70 into the DC. These findings reveal a novel mechanism by which tTregs can maintain tolerance or prevent excessive, proinflammatory Th1 responses.     

Oral immunization of mice with Saccharomyces cerevisiae expressing a neutralizing epitope of ApxIIA exotoxin from Actinobacillus pleuropneumoniae induces systemic and mucosal immune responses

An oral delivery system based on ApxIIA#5‐expressed on Saccharomyces cerevisiae was studied for its potential to induce immune responses in mice. Murine bone marrow‐derived dendritic cells (DCs) stimulated in vitro with ApxIIA#5‐expressed on S. cerevisiae upregulated the expression of maturation and activation markers, leading to production of tumor necrosis factor‐α, interleukin (IL)‐1β, IL‐12p70 and IL‐10. Presentation of these activated DCs to cluster of differentiation CD4+ T cells collected from mice that had been orally immunized with the ApxIIA#5‐expressed on S. cerevisiae elicited specific T‐cell proliferation. In addition, the orally immunized mice had stronger antigen‐specific serum IgG and IgA antibody responses and larger numbers of antigen‐specific IgG and IgA antibody‐secreting cells in their spleens, Peyer's patches and lamina propria than did those immunized with vector‐only S. cerevisiae or those not immunized. Furthermore, oral immunization induced T helper 1‐type immune responses mediated via increased serum concentrations of IgG2a and an increase predominantly of IFN‐γ‐producing cells in their spleens and lamina propria. Our findings suggest that surface‐displayed ApxIIA#5‐expressed on S. cerevisiae may be a promising candidate for an oral vaccine delivery system for eliciting systemic and mucosal immunity.     

Dendritic cells engineered to express CD40L continuously produce IL12 and resist negative signals from Tr1/Th3 dominated tumors

TNFα-matured dendritic cells (DCs) pulsed with tumor antigens are being evaluated as cancer vaccines. It has been shown that DCs produce IL12 during a limited time span and subsequently enter a stage of IL12 exhaustion. If DCs are generated ex vivo, the patient could receive IL12-exhausted DCs which may be detrimental for stimulating anti-tumor Th1 responses. Furthermore, many cancer patients exhibit a cytokine profile skewed toward IL10 and TGFβ. This immunological profile, called the Tr1/Th3 response, is associated with the presence of regulatory T-cells. Tr1/Th3 responses potently inhibit DC maturation, thereby regulating Th1 responses. In the present study, we produced genetically engineered DCs that continuously express Th1-related cytokines such as IL12, and resist negative signals from Tr1/Th3-dominated bladder carcinoma cells. Human immature DCs were genetically engineered by adenoviral vectors to express CD40L, or were treated with TNFα as a positive control for maturation. The expression of different Th1/Th3 and inflammatory cytokines was monitored. IL12 and IFNγ were expressed by CD40L-engineered DCs, while TNFα-matured DCs lacked IFNγ and exhibited low IL12 expression. The addition of recombinant IL10 to genetically engineered DCs did not abolish their Th1 profile. Likewise, coculture with tumor cell lines expressing TGFβ with or without recombinant IL10 did not revert to the engineered DCs. We further demonstrate that the resistance of CD40L-expressing DCs to TGFβ and IL10 may be due to decreased levels of TGFβ and IL10 receptors. Thus, CD40L-engineered DCs are robust Th1-promoting ones that are resistant to Tr1/Th3-signaling via IL10 and TGFβ.     

Oncostatin M induces dendritic cell maturation and Th1 polarization

Oncostatin M (OSM) is a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family that has been found to be involved in both pro- and anti-inflammatory responses in cell-mediated immunity. Maturation of dendritic cells (DCs) is crucial for initiation of primary immune responses and is regulated by several stimuli. In this study, the role of OSM in the phenotypic and functional maturation of DCs was evaluated in vitro. Stimulation with OSM upregulated the expression of CD80, CD86, MHC class I and MHC class II and reduced the endocytic capacity of immature DCs. Moreover, OSM induced the allogeneic immunostimulatory capacity of DCs by stimulating the production of the Th1-promoting cytokine IL-12. OSM also increased the production of IFN-γ by T cells in mixed-lymphocyte reactions, which would be expected to contribute to the Th1 polarization of the immune response. The expression of surface markers and cytokine production in DCs was mediated by both the MAPK and NF-κB pathways. Taken together, these results indicate that OSM may play a role in innate immunity and in acquired immunity by enhancing DCs maturation and promoting Th1 immune responses.     

miR‐181a and miR‐150 regulate dendritic cell immune inflammatory responses and cardiomyocyte apoptosis via targeting JAK1–STAT1/c‐Fos pathway

The immune inflammatory response plays a crucial role in many cardiac pathophysiological processes, including ischaemic cardiac injury and the post‐infarction repair process. MicroRNAs (miRNAs) regulate the development and function of dendritic cells (DCs), which are key players in the initiation and regulation of immune responses; however, the underlying regulatory mechanisms remain unclear. Here, we used the supernatants of necrotic primary cardiomyocytes (Necrotic‐S) to mimic the myocardial infarction (MI) microenvironment to investigate the role of miRNAs in the regulation of DC‐mediated inflammatory responses. Our results showed that Necrotic‐S up‐regulated the DC maturation markers CD40, CD83 and CD86 and increased the production of inflammatory cytokines, concomitant with the up‐regulation of miR‐181a and down‐regulation of miR‐150. Necrotic‐S stimulation activated the JAK/STAT pathway and promoted the nuclear translocation of c‐Fos and NF‐κB p65, and silencing of STAT1 or c‐Fos suppressed Necrotic‐S‐induced DC maturation and inflammatory cytokine production. The effects of Necrotic‐S on DC maturation and inflammatory responses, its activation of the JAK/STAT pathway and the induction of cardiomyocyte apoptosis under conditions of hypoxia were suppressed by miR‐181a or miR‐150 overexpression. Taken together, these data indicate that miR‐181a and miR‐150 attenuate DC immune inflammatory responses via JAK1–STAT1/c‐Fos signalling and protect cardiomyocytes from cell death under conditions of hypoxia.     

Is there a role for mannan-binding lectin in the diagnosis of inflammatory bowel disease?

Mannan-binding lectin (MBL) activates the lectin-complement pathway as part of the innate immune defence by binding to the surface of microorganisms. Therefore, MBL2 presents an interesting candidate gene for the inflammatory bowel diseases, ulcerative colitis (UC) and Crohn's disease (CD). In our study, we evaluated the MBL serum concentrations and genotypes for diagnostic and classification purposes of patients with CD and UC. The MBL serum concentration was analysed in 98 CD patients and in 83 UC patients. In total, 82 patients with inflammatory rheumatic disorders and 189 healthy individuals served as controls. All study subjects were genotyped for the MBL2 polymorphisms G54D, G57E and R52C and the NOD2 (CARD15) mutations R702W, G908R and L1007fsinsC. Neither the median MBL serum concentration nor the MBL2 genotype distribution differed significantly between cohorts. Measurement of MBL serum concentrations offers no benefit for the diagnosis of CD or UC.     

Ficolins and infectious diseases

Ficolins are serum complement lectins, with a structure similar to mannose-binding lectin (MBL) and lung surfactant protein (SP)-A and SP-D. Ficolins activate the lectin complement system and play important roles in host innate immunity. Ficolins are members of the collectin family of proteins, which act as pattern recognition receptors (PRRs). They are soluble oligomeric defense proteins with lectin-like activity, and are able to recognize pathogen-associated molecular patterns (PAMPs), which are carbohydrate molecules on the surface of pathogens, and of apoptotic, necrotic, and malignant cells. Upon binding to their specific PAMPs, ficolins may trigger activation of the immune system either (1) by initiating activation of complement via the lectin pathway, (2) by a primitive type of opsonophagocytosis, or (3) by stimulating secretion of the inflammatory cytokines interferon (IFN)-Γ, interleukin (IL)-17, IL-6, and tumor necrosis factor (TNF)-α, and production of nitric oxide (NO) by macrophages, thus limiting the infection and concurrently orchestrating the subsequent adaptive immune response. Recently, a number of reports have shown that dysfunction or abnormal expression of ficolins may play crucial roles in viral and bacterial diseases and in inflammation. This review summarizes the reports on the roles of ficolins in the infectious diseases, and provides insight into ficolins as novel innate immune therapeutic options to treat these diseases.     

RNA-containing adenovirus/polyethylenimine transfer complexes effectively transduce dendritic cells and induce antigen-specific T cell responses

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells in initiating primary immune responses. Given the unique properties of DCs, gene-modified DCs represent a particularly attractive approach for immunotherapy of diseases such as cancer. METHODS: Gene-modified DCs were obtained by a receptor-mediated gene delivery system using adenovirus (Ad) particles as ligand and RNA or DNA condensed by polyethylenimine (PEI). In vitro transcribed polyadenylated or non-polyadenylated RNA was used. RNA-transduced DCs were generated expressing chicken ovalbumin (OVA) or chimeric constructs thereof, and compared with DNA-transduced DCs. RESULTS: Ad/PEI transfection complexes efficiently delivered RNA into DCs. Such RNA-transduced DCs induced OVA-specific T cell responses more effectively than DNA-transduced DCs. Furthermore, DCs transduced with polyadenylated RNA were more potent in stimulating CD4(+) and CD8(+) T cell responses than DCs transduced with non-polyadenylated RNA and this was particularly important for CD4(+) T cell responses. CONCLUSIONS: Ad/PEI/RNA transfection is an efficient means for generating RNA-transduced DCs and for stimulating antigen-specific T cell responses. Polyadenylation of RNA enhances CD8(+) T cell responses and is essential for CD4(+) T cell responses.     

Cytokine and cellular responses to human herpesvirus‐6B in patients with B‐acute lymphoblastic leukemia

Primary infection with human herpesvirus‐6 (HHV‐6), is followed by its lifelong persistence in the host. Most T‐cell responses to HHV‐6 have been characterized using peripheral blood from healthy adults; however, the role of HHV‐6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV‐6 infection in patients with B‐acute lymphoblastic leukemia (B‐ALL) was analyzed. HHV‐6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti‐ and pro‐inflammatory cytokines (IL‐4, IL‐1, IL‐6, IL‐8, IL‐12p70, IL‐17a, TNF‐α and IFN‐γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV‐6 virus loads in blood. Characterization of T‐cell responses to HHV‐6 showed low specific T‐cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN‐γ‐producing T cells were detected in 0.03%–0.23% and in 0%–0.2% of CD4+T cells, respectively. Strong production of IL‐6 was detected in medium supernatants of challenged T‐cells whatever the HHV‐6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF‐α and IFN‐γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV‐6 in blood was identified, suggesting that HHV‐6 is not strongly associated with development of B‐ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV‐6B specific immune responses may be below the detection threshold of the assays used.     

CC-chemokine ligand 16 induces a novel maturation program in human immature monocyte-derived dendritic cells

Dendritic cells (DCs) are indispensable for initiation of primary T cell responses and a host's defense against infection. Many proinflammatory stimuli induce DCs to mature (mDCs), but little is known about the ability of chemokines to modulate their maturation. In the present study, we report that CCL16 is a potent maturation factor for monocyte-derived DCs (MoDCs) through differential use of its four receptors and an indirect regulator of Th cell differentiation. MoDCs induced to mature by CCL16 are characterized by increased expression of CD80 and CD86, MHC class II molecules, and ex novo expression of CD83 and CCR7. They produce many chemokines to attract monocytes and T cells and are also strong stimulators in activating allogeneic T cells to skew toward Th1 differentiation. Interestingly, they are still able to take up Ag and express chemokine receptors usually bound by inflammatory ligands and can be induced to migrate to different sites where they capture Ags. Our findings indicate that induction of MoDC maturation is an important property of CCL16 and suggest that chemokines may not only organize the migration of MoDCs, but also directly regulate their ability to prime T cell responses.     

Modulation of the development of human monocyte‐derived dendritic cells by lithium chloride

Lithium has been used or explored to treat psychiatric and neurodegenerative diseases that are frequently associated with an abnormal immune status. It is likely that lithium may work through modulation of immune responses in these patients. Because dendritic cells (DC) play a central role in regulating immune responses, this study investigated the influence of lithium chloride (LiCl) on the development and function of DC. Exposure to LiCl during the differentiation of human monocyte‐derived immature DCs (iDC) enhances CD86 and CD83 expression and increases the production of IL‐1β, IL‐6, IL‐8, IL‐10, and TNF‐α. However, the presence of LiCl during LPS‐induced maturation of iDC has the opposite effect. During iDC differentiation, LiCl suppresses the activity of glycogen synthase kinase (GSK)‐3β, and activates PI3K and MEK. In addition, LiCl activates peroxisome proliferator‐activated receptor γ (PPARγ) during iDC differentiation, a pathway not described before. Each of these signaling pathways appears to have distinct impact on the differentiating iDC. The enhanced CD86 expression by LiCl involves the PI3K/AKT and GSK‐3β pathway. LiCl modulates the expression of CD83 in iDC mainly through MEK/ERK, PI3K/AKT, and PPARγ pathways, while the increased production of IL‐1β and TNF‐α mainly involves the MEK/ERK pathway. The effect of LiCl on IL‐6/IL‐8/IL‐10 secretion in iDC is mediated through inhibition of GSK‐3β. We have also demonstrated that PPARγ is downstream of GSK‐3β and is responsible for the LiCl‐mediated modulation of CD86/83 and CD1 expression, but not IL‐6/8/10 secretion. The combined influence of these molecular signaling pathways may account for certain clinical effect of lithium. J. Cell. Physiol. 226: 424–433, 2011. © 2010 Wiley‐Liss, Inc.