Prostaglandin E2-dependent IL-23 production in aged murine dendritic cells

CD4+ T cells of the Th17 subtype are over-represented in the aged immune system. Dendritic cells (DC) play a critical role in naïve CD4+ T cell differentiation. However, expression of cytokines by aged DC that promote differentiation or survival of Th17 cells has not been extensively investigated. Using bone marrow-derived DC from C57BL/6 mice of different ages we compared cytokine production after DC activation by Toll-like receptor agonists for TLR4 and/or TLR7/8. DC-derived TNF-α and IL-12p70 production and expression of DC co-stimulatory molecules did not vary significantly by age indicating that TLR expression, function and signal transduction were intact in aged DC. There were relatively minor age-related changes in TGF-β and IL-6 which promote Th17 differentiation, but IL-23, a Th17-suvival cytokine, increased more than 40-fold across the lifespan. DC-derived prostaglandin E2 (PGE2) also increased with age and the up-regulation of IL-23 expression by aged DC was blocked by indomethacin that prevents PGE2 production, and by antagonists of PGE2 receptors. Exogenous PGE2 added to DC cultures further enhanced IL-23 production from aged but not young DCs. These data indicate that age-related changes in DC PGE2 production are necessary, but not sufficient to induce DC IL-23 production. Such changes may play a role in the expansion of Th17 cells in the aged immune system.     

Interleukin-18 attracts plasmacytoid dendritic cells (DC2s) and promotes Th1 induction by DC2s through IL-18 receptor expression

In vivo evidence suggests that interleukin-18 (IL-18) shapes the development of adaptive immunity toward T-helper cell type 1 (Th1) responses. Monocyte-derived dendritic cells 1 (DC1s) preferentially induce a Th1 response, while plasmacytoid DC-derived DC2s have been linked to a Th2 response. We analyzed the role of IL-18 during the initiation phase of a Th response in vitro to elucidate the basis of these in vivo observations. IL-18 was constitutively released from DC1s, but not DC2s. Neutralization of IL-18 in coculture experiments of DC1s with allogeneic naive T lymphocytes did not alter the Th1/Th2 phenotype, while anti-IL-12 efficiently down-regulated the Th1 response. Unexpectedly, IL-18 receptor (IL-18R) alpha and beta chains were expressed on DC2 lineage. IL-18R expression was functional, as IL-18 induced chemotaxis in plasmacytoid DCs (pre-DC2s) and enhanced the allostimulatory capacity of IL-3-differentiated DC2s. Pre-DC2s exposed to IL-18 skewed the development of Th cells toward Th1 in coculture experiments of DC2s and allogeneic naive T cells, which was inhibited by IL-12 p70 neutralization. IL-18 might have a profound role during the initiation phase of an immune response by recruiting pre-DC2s and modulating the function of DC2s.     

Interleukin-21 inhibits dendritic cell activation and maturation

Interleukin 21 (IL-21) is a newly described cytokine with homology to IL-4 and IL-15. They belong to a cytokine family that uses the common gamma chain for signaling but also have their private high-affinity receptors. Since it is well known that IL-4 modulates differentiation and activation of dendritic cells (DCs), we analyzed effects of IL-21 compared with IL-15 on DC differentiation, maturation, and function. Here we show that DCs generated with granulocyte-macrophage colony-stimulating factor (GMCSF) in the presence of IL-21 (IL-21DCs) differentiated into phenotypically and functionally altered DCs characterized by reduced major histocompatibility complex class II (MHCII) expression, high antigen uptake, and low stimulatory capacity for T-cell activation in vitro. Additionally, IL-21DCs completely failed to induce antigen (Ag)-specific T-cell mediated contact hypersensitivity. Furthermore, IL-21 blocked lipopolysaccharide (LPS)-induced activation and maturation of DCs, which was not mediated by release of the anti-inflammatory cytokine IL-10. In contrast, when supplementing GMCSF with IL-15, DCs differentiated into mature antigen-presenting cells (APCs) with low antigen uptake and highly significant increased capacities to stimulate T cells in vitro and in vivo. Taken together, these results identify a dichotomous action of these structurally related cytokines on DCs, establishing IL-21 as inhibitory cytokine on DC activation and IL-15 as potent stimulator of DC function, making both cytokines interesting targets for therapeutic manipulation of DC-induced immune reactions.     

Aging affects AO rat splenic conventional dendritic cell subset composition, cytokine synthesis and T-helper polarizing capacity

It is well-established that almost all cellular components of innate and adaptive immunity undergo age-related remodelling. The findings on age-related changes in both human and mouse dendritic cells (DCs) are conflicting, whereas there are no data on the influence of aging on rat DCs. In an attempt to fill this gap, freshly isolated splenic DCs expressing CD103 (α_OX-62 integrin), a DC specific marker recognized by MRC OX62 monoclonal antibody, from 3- (young) and 26-month-old (aged) Albino Oxford rats were examined for subset composition, expression of activation/differentiation markers (CD80, CD86 and CD40 and MHC II molecules) and endocytic capacity using flow cytometric analysis (FCA). In addition, splenic OX62+ DCs cultured in the presence or absence of LPS were analysed for the activation marker and TNF-α, IL-6, IL-12, IL-23, TGF-β1, IL-10 expression using FCA, RT-PCR and ELISA, respectively. Moreover, the allostimulatory capacity of OX62+ DCs and IFN-γ, IL-4 and IL-17 production by CD4+ T cells in mixed leukocyte reaction was quantified using FCA and ELISA, respectively. It was found that aging: i) shifts the CD4+:CD4? subset ratio in the OX62+ DCs population towards the CD4? subset and ii) influences DCs maturation (judging by activation marker expression and efficiency of endocytosis) by affecting the expression of intrinsic (TNF-α and IL-10) and extrinsic maturation regulators. Furthermore, in LPS-matured OX62+ DCs from aged rats expression of TNF-α, IL-12, IL-23 and IL-6 was increased, whereas that of IL-10 was diminished compared with the corresponding cells from young rats. Moreover, in MLR, OX62+ DCs from aged rats exhibited enhanced Th1/Th17 driving force and diminished allostimulatory capacity compared with those from young rats.     

Hepatic environment elicits monocyte differentiation into a dendritic cell subset directing Th2 response

BACKGROUND/AIMS: Dendritic cells (DCs), which play a critical role during immune response, could present alternative differentiation patterns depending on tissue microenvironment. Our aim was to examine the influence of hepatic microenvironment on human monocyte differentiation into DCs. METHODS: Cytology, immunophenotyping, cytokine production and T-cell activation were analyzed in DCs differentiated from human monocytes co-cultured with rat liver epithelial cells (RLEC) or human cells from various tissue origins and compared to control DCs obtained on plastic with GM-CSF/IL-4. RESULTS: RLEC environment promotes DC differentiation in the presence of IL-4 without GM-CSF. These DCs evidence similar expression of MHC-II, co-stimulatory and adhesion molecules than control DCs, but distinct lineage markers defining a CD11c+/CD14+/CD123+ DC subset. This phenotype is common to DCs from RLEC and human liver environment and differs from that obtained with skin or intestine environments. Functionally, they produce IL-10 but not IL-12p70 and favor IL-4/IL-10 secretion by T-cells rather than IFN-gamma. CONCLUSIONS: Our results confirm that tissue niches modulate DC differentiation and demonstrate that hepatic environment influences monocyte differentiation into a DC subset directing Th2 response, a key data for understanding the specialized immune response in liver. They also make RLEC co-culture system useful for studying liver DC functions.     

Fasciola hepatica tegumental antigens indirectly induce an M2 macrophage‐like phenotype in vivo

The M2 subset of macrophages has a critical role to play in host tissue repair, tissue fibrosis and modulation of adaptive immunity during helminth infection. Infection with the helminth, Fasciola hepatica, is associated with M2 macrophages in its mammalian host, and this response is mimicked by its excretory‐secretory products (FhES). The tegumental coat of F. hepatica (FhTeg) is another major source of immune‐modulatory molecules; we have previously shown that FhTeg can modulate the activity of both dendritic cells and mast cells inhibiting their ability to prime a Th1 immune response. Here, we report that FhTeg does not induce Th2 immune responses but can induce M2‐like phenotype in vivo that modulates cytokine production from CD4⁺ cells in response to anti‐CD3 stimulation. FhTeg induces a RELMα expressing macrophage population in vitro, while in vivo, the expression of Arg1 and Ym‐1/2 but not RELMα in FhTeg‐stimulated macrophages was STAT6 dependent. To support this finding, FhTeg induces RELMα expression in vivo prior to the induction of IL‐13. FhTeg can induce IL‐13‐producing peritoneal macrophages following intraperitoneal injection This study highlights the important role of FhTeg as an immune‐modulatory source during F. hepatica infection and sheds further light on helminth–macrophage interactions.     

Mammalian target of rapamycin and glycogen synthase kinase 3 differentially regulate lipopolysaccharide-induced interleukin-12 production in dendritic cells

Phosphoinositide 3-kinase (PI3K) negatively regulates Toll-like receptor (TLR)–mediated interleukin-12 (IL-12) expression in dendritic cells (DCs). We show here that 2 signaling pathways downstream of PI3K, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 (GSK3), differentially regulate the expression of IL-12 in lipopolysaccharide (LPS)–stimulated DCs. Rapamycin, an inhibitor of mTOR, enhanced IL-12 production in LPS-stimulated DCs, whereas the activation of mTOR by lentivirus-mediated transduction of a constitutively active form of Rheb suppressed the production of IL-12. The inhibition of protein secretion or deletion of IL-10 cancelled the effect of rapamycin, indicating that mTOR regulates IL-12 expression through an autocrine action of IL-10. In contrast, GSK3 positively regulates IL-12 production through an IL-10–independent pathway. Rapamycin-treated DCs enhanced Th1 induction in vitro compared with untreated DCs. LiCl, an inhibitor of GSK3, suppressed a Th1 response on Leishmania major infection in vivo. These results suggest that mTOR and GSK3 pathways regulate the Th1/Th2 balance though the regulation of IL-12 expression in DCs. The signaling pathway downstream of PI3K would be a good target to modulate the Th1/Th2 balance in immune responses in vivo.     

Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype

Osteopontin (OPN) has been shown to have T helper 1 (Th1) cytokine functions in cell-mediated immunity. Deficiency of OPN is linked to a reduced Th1 immune response in autoimmunity, infectious disease, and delayed-type allergy. Dendritic cells (DCs) are central for the induction of T-cell-mediated immunity, when initially flexible DCs are instructed by priming signals and tissue-derived factors to adopt Th1, Th2, or regulatory T-cell-inducing phenotypes. Although OPN influences the cytokine secretion of T cells and macrophages, its effects on DC polarization remain an important missing link in the understanding of OPN functions in Th1 immunity. Here we demonstrate that OPN promotes the emigration of human DCs from the epidermis and functionally activates myeloid-type DCs, augmenting their expression of HLA-DR, costimulatory, and adhesion molecules. OPN induces their Th1-promoting tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) secretion, and enhances their allostimulatory capacity. In mixed lymphocyte reactions (MLRs), OPN stimulates IL-12 secretion by DCs, inducing elevated interferon-gamma (IFN-gamma) production by T cells. Naive Th cells stimulated by OPN-activated DCs show a Th1-polarized cytokine production. Our findings identify OPN as an important tissue-derived factor that DCs encounter when traveling from peripheral sites of activation to secondary lymphatic organs, which induces DC maturation toward a Th1-promoting phenotype.     

Dendritic cell-derived IL-2 production is regulated by IL-15 in humans and in mice

Dendritic cells (DCs) are involved in the initiation and regulation of innate and adaptive immune responses. Several molecular mechanisms regulate these diverse DC functions, and we have previously reported that mouse dendritic cells (mDCs) can produce interleukin-2 (IL-2) in vitro and in vivo, in response to microbial activation and T-cell-mediated stimuli. This property is shared by different DC subtypes, including Langerhans cells. Here we show that, on appropriate stimulation, human DCs, both plasmacytoid and myeloid subtypes, also express IL-2. Interestingly, the production of IL-2 by myeloid DCs is induced by T-cell-mediated stimuli and depends on the presence of IL-15. The key role of this cytokine in regulating IL-2 production was also confirmed in the mouse system. In particular, we could show that DCs from IL-15-deficient mice were strongly impaired in the ability to produce IL-2 after interactions with different microbial stimuli. Our results indicate that DC-produced IL-2 is tightly coregulated with the expression of IL-15.     

Regulatory dendritic cells program generation of interleukin-4-producing alternative memory CD4 T cells with suppressive activity

The heterogeneity and mechanisms for the generation of CD4 memory T (CD4 Tm) cells remain elusive. Distinct subsets of dendritic cells (DCs) have been found to regulate a distinct T-helper (Th)-cell subset differentiation by influencing cytokine cues around CD4 T cells; however, whether and how the regulatory DC subset can regulate Tm-cell differentiation remains unknown. Further, there is no ideal in vitro experimental system with which to mimic the 3 phases of the CD4 T-cell immune response (expansion, contraction, memory generation) and/or to culture CD4 Tm cells for more than a month. By analyzing CD4 T cells programmed by long-term coculture with regulatory DCs, we identified a population of long-lived CD4 T cells with a CD44(hi)CD62L(-)CCR7(-) effector memory phenotype and rapid, preferential secretion of the Th2 cytokines interleukin-4 (IL-4), IL-5, IL-10, and IL-13 after antigenic stimulation. These regulatory DC-programmed Tm cells suppress CD4 T-cell activation and proliferation in vitro via IL-10 and inhibit the delayed-type hypersensitivity response once infused in vivo. We also identify their natural counterpart, which is up-regulated by regulatory DC transfusion and negatively regulates the recall response in vivo. Different from interferon-γ-producing conventional Tm cells, these IL-4-producing CD4 Tm cells act as alternative Tm cells with a regulatory function, suggesting a new way of negative immune regulation by memory T cells.     

Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10

Dendritic cell (DC) activation through CD40-CD40 ligand interactions is a key regulatory step for the development of protective T-cell immunity and also plays an important role in the initiation of T-cell responses involved in autoimmune diseases and allograft rejection. In contrast to previous reports, we show that the immunosuppressive drug dexamethasone (DEX) redirects rather than simply blocks this DC activation process. We found that DCs triggered through CD40 in the presence of DEX were unable to acquire high levels of costimulatory, adhesion, and major histocompatibility complex class I and II molecules and failed to express the maturation marker CD83, whereas antigen uptake was not affected. Moreover, DEX strikingly modified the CD40-activated DC cytokine secretion profile by suppressing the production of the proinflammatory cytokine interleukin (IL)-12 and potentiating the secretion of the anti-inflammatory cytokine IL-10. Accordingly, DEX-exposed CD40-triggered DCs displayed a decreased T-cell allostimulatory potential and a dramatically impaired ability to activate cloned CD4(+) T helper 1 (Th1) cells. Moreover, interaction between Th1 cells and these DCs rendered the T cells hyporesponsive to further antigen-specific restimulation. Collectively, our results demonstrate that DEX profoundly modulates CD40-dependent DC activation and suggest that the resulting alternatively activated DCs can be exploited for suppression of unwanted T-cell responses in vivo.     

Synergistic activation of dendritic cells by combined Toll-like receptor ligation induces superior CTL responses in vivo

Toll-like receptors (TLRs) are able to interact with pathogen-derived products and their signals induce the coordinated activation of innate and adaptive immune mechanisms. Dendritic cells (DCs) play a central role in these events. As the different TLRs are able to trigger MyD88/TRIF-dependent and -independent signaling pathways, we wondered if the simultaneous activation of these signaling cascades would synergize with respect to DC activation and induce superior cytotoxic T-lymphocyte (CTL) activity in vivo. We observed that indeed the combined activation of MyD88-dependent and -independent signaling induced by TLR7 and TLR3 ligands provoked a more rapid and more sustained bone marrow-derived DC (BMDC) activation with regard to the secretion of proinflammatory cytokines, like IL-6 and IL-12p70, and the expression of costimulatory molecules like CD40, CD70, and CD86. Furthermore, in the presence of combined TLR ligand-stimulated DCs, CD4(+) and CD8(+) T cells were insensitive toward the inhibitory effects of regulatory T cells. Most importantly, peptide-loaded BMDCs stimulated by TLR ligand combinations resulted in a marked increase of CTL effector functions in wild-type mice in vivo. Thus, our results provide evidence that unlocking the full potential of DCs by advanced activation protocols will boost their immunogenic potential and improve DC-based vaccination strategies.     

Natural-killer cells and dendritic cells: "l'union fait la force"

Several recent publications have focused on the newly described interactions between natural-killer (NK) cells and dendritic cells (DCs). Activated NK cells induce DC maturation either directly or in synergy with suboptimal levels of microbial signals. Immature DCs appear susceptible to autologous NK-cell-mediated cytolysis while mature DCs are protected. NK-cell-induced DC activation is dependent on both tumor necrosis factor-alpha (TNF-alpha)/interferon-gamma (IFN-gamma) secretion and a cell-cell contact involving NKp30. In vitro, interleukin-12 (IL-12)/IL-18, IL-15, and IFN-alpha/beta production by activated DCs enhance, in turn, NK-cell IFN-gamma production, proliferation, and cytotoxic potential, respectively. In vivo, NK-cell/DC interactions may occur in lymphoid organs as well as in nonlymphoid tissues, and their consequences are multiple. By inducing DC activation, NK-cell activation induced by tumor cells can indirectly promote antitumoral T-cell responses. Reciprocally, DCs activated through Toll-like receptors (TLRs) induce potent NK-cell activation in antiviral responses. Thus, DCs and NK cells are equipped with complementary sets of receptors that allow the recognition of various pathogenic agents, emphasizing the role of NK-cell/DC crosstalk in the coordination of innate and adaptive immune responses.     

CD40–CD40L cross-talk integrates strong antigenic signals and microbial stimuli to induce development of IL-17-producing CD4+ T cells

IL-17-producing CD4⁺ T cells have been recognized as key players in organ-related autoimmune disease; however, the parameters that govern their development are yet to be elucidated fully. By using both in vivo and in vitro systems, we have investigated the role of antigen dose, pathogen-associated molecular patterns, and CD40–CD40 ligand (CD40L) cross-talk in Th17 differentiation. We found that the strength of antigenic stimulation critically influenced the extent of Th17 differentiation, because high, but not low or intermediate, antigen concentrations led to IL-17 production. Strong antigenic stimulation of T cells up-regulated CD40L expression, which in concert with certain microbial stimuli (i.e., cytosine phosphate guanine, curdlan, and zymosan) synergistically increased dendritic cell (DC) IL-6 production and Th17 polarization. CD40-deficient DCs exhibited reduced cytokine release and failed to drive Th17 development in vitro. These results were confirmed in vivo where the absence of CD40–CD40L cross-talk was found to prevent the expansion of IL-17-producing cells and accordingly the development of experimental autoimmune encephalitis. Our data demonstrate that CD40–CD40L cross-talk is important for Th17 development by translating strong T cell receptor and microbial stimuli into IL-6 production.     

NF－κB activation and zinc finger protein A20 expression in mature dendritic cells derived from liver allografts undergoing acute rejection

AIM：To investigate the role of NF-κB activation and zinc finger protein A20 expression in the regulation of maturation of dendritic cells (DCs) derived from liver allografts undergoing acute acute rejection.METHODS:Sixty donor male SD rats and sixty recipient male lew rats weighing 220-300 g were randomly divided into whole liver transplantation group and partial liver transplantation group. Allogeneic (SD rat to LEW rat) whole and 50% partial liver transplantation were performed. DCs from liver grafts 0 hour and 4 days after transplantation were isolated and propagated in the presence of GM-CSF in vitro. Morphological characteristics and phenotypical features of DCs propagated for 10 days were analyzed by electron microscopy and flow cytometry,respectively. NF-κB binding activity, IL-1 2P70 Protein and zinc finger protein A20 expression on these DCs were measured by EMSA and Western blotting, respectively. Histological grading of rejection was determined.RESULTS: Allogeneic whole liver grafts showed no signs of rejection on day 4 after the transplantation. In contrast,allogeneic partial liver grafts demonstrated moderate to severe rejection on day 4 after the transplantation. After propagation for 10 days in the presence of GM-CSF in vitro,DCs from allogeneic whole liver grafts exhibited features of immature DC with absence of CD40 surface expression,these DCs were found to exhibit detectable but very low level of NF-κB activity, 1L-12 p70 protein and zinc finger protein A20 expression. Whereas, DCs from allogeneic partial liver graft 4 days after transplantation displayed features of mature DC, with high level of CD40 surface expression, and as a consequence, higher expression of lL-12p70 protein, higher activities of NF-κB and higher expression of zinc finger protein A20 compared with those of DCs from whole liver grafts (P<0.001).CONCLUSION: These results suggest that A20 expression is up-regulated in response to NF-κB activation in mature DCs derived from allogeneic liver grafts undergoing acute rejection. Given the NF-κB inhibition function of this gene, it is suggested that their expression survives to limit NF-κB activation and maturation of DCs,and consequently inhibits the acute rejection and induces acceptance of liver graft.     

NP30 stimulates Th17 differentiation through DC in Schistosomiasis Japonicum

The murine monoclonal anti‐idiotypic antibody, NP30, is a potential vaccine candidate against Schistosoma japonicum. Previous studies have revealed that NP30 has an immunoregulatory effect, but the underlying mechanism for this effect remains unknown. This study shows that NP30 induces dendritic cell (DC) maturation and increases the production of pro‐inflammatory cytokines. The expression of CD86 and MHC II was upregulated in DCs following stimulation with NP30 in vitro. Moreover, NP30 induced Th17 polarization by increasing the production of IL‐6 and TGF‐β. In vivo, Th17 differentiation was induced by the production of key pro‐inflammatory cytokines, including IL‐6and TGF‐β, from DCs of NP30‐immunized mice. These results indicate that NP30 promotes Th17 polarization through DC activation, preventing serious schistosomiasis.