Dendritic cells post-maturation are reprogrammed with heightened IFN-gamma and IL-10

Mature dendritic cells (mDCs) undergo "exhaustion" in producing cytokines. Nevertheless, whether this "exhaustion" of mDCs is selective to certain cytokines, or whether mDCs have specific cytokine-producing profiles has yet to be defined. Herein, we investigated the cytokine production in vitro by immature DCs (iDCs) and LPS-induced mDCs. Compared to iDCs, mDCs produced comparable levels of IL-6 and TNF-alpha. Strikingly, mDCs produced significantly higher IFN-gamma and IL-10. IL-12 production of mDCs was suppressed. Kinetic studies of the responses of iDCs and mDCs to LPS or CD40L showed that mDCs acquired progressively heightened activity in producing IFN-gamma and IL-10. TNF-alpha-, IL-6-producing capability of mDCs was maintained. Nevertheless, IL-12 production by mDCs was not recovered at any time point. Mature DCs were potent in priming both Th1 and Th2 cells. In conclusion, upon maturation, DCs are reprogrammed with a distinct cytokine-secreting profile, which may play an important role in regulating T cell functions.     

Dendritic cell maturation requires STAT1 and is under feedback regulation by suppressors of cytokine signaling

In this study we show that activation of STAT pathways is developmentally regulated and plays a role in dendritic cell (DC) differentiation and maturation. The STAT6 signaling pathway is constitutively activated in immature DC (iDC) and declines as iDCs differentiate into mature DCs (mDCs). However, down-regulation of this pathway during DC differentiation is accompanied by dramatic induction of suppressors of cytokine signaling 1 (SOCS1), SOCS2, SOCS3, and cytokine-induced Src homology 2-containing protein expression, suggesting that inhibition of STAT6 signaling may be required for DC maturation. In contrast, STAT1 signaling is most robust in mDCs and is not inhibited by the up-regulated SOCS proteins, indicating that STAT1 and STAT6 pathways are distinctly regulated in maturing DC. Furthermore, optimal activation of STAT1 during DC maturation requires both IL-4 and GM-CSF, suggesting that synergistic effects of both cytokines may in part provide the requisite STAT1 signaling intensity for DC maturation. Analyses of STAT1(-/-) DCs reveal a role for STAT1 in repressing CD86 expression in precursor DCs and up-regulating CD40, CD11c, and SOCS1 expression in mDCs. We further show that SOCS proteins are differentially induced by IL-4 and GM-CSF in DCs. SOCS1 is primarily induced by IL-4 through a STAT1-dependent mechanism, whereas SOCS3 is induced mainly by GM-CSF. Taken together, these results suggest that cytokine-induced maturation of DCs is under feedback regulation by SOCS proteins and that the switch from constitutive activation of the STAT6 pathway in iDCs to predominant use of STAT1 signals in mDC is mediated in part by STAT1-induced SOCS expression.     

CD47 engagement inhibits cytokine production and maturation of human dendritic cells

Upon encounter with bacterial products, immature dendritic cells (iDCs) release proinflammatory cytokines and develop into highly stimulatory mature DCs. In the present study, we show that human monocyte-derived DCs functionally express the CD47 Ag, a thrombospondin receptor. Intact or F(ab')2 of CD47 mAb suppress bacteria-induced production of IL-12, TNF-alpha, GM-CSF, and IL-6 by iDCs. 4N1K, a peptide derived from the CD47-binding site of thrombospondin, also inhibits cytokine release. The inhibition of IL-12 and TNF-alpha is IL-10-independent inasmuch as IL-10 production is down-modulated by CD47 mAb and blocking IL-10 mAb fails to restore cytokine levels. CD47 ligation counteracts the phenotypic and functional maturation of iDCs in that it prevents the up-regulation of costimulatory molecules, the loss of endocytic activity, and the acquisition of an increased capacity to stimulate T cell proliferation and IFN-gamma production. Interestingly, regardless of CD47 mAb treatment during DC maturation, mature DC restimulated by soluble CD40 ligand and IFN-gamma, to mimic DC/T interaction, produce less IL-12 and more IL-18 than iDCs. Finally, CD47 ligation on iDCs does not impair their capacity to phagocytose apoptotic cells. We conclude that following exposure to microorganisms, CD47 ligation may limit the intensity and duration of the inflammatory response by preventing inflammatory cytokine production by iDCs and favoring their maintenance in an immature state.     

Functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells

Dendritic cells (DCs) potently stimulate the transmission of human immunodeficiency virus type 1 (HIV-1) to CD4(+) T cells. Immature DCs (iDCs) located in submucosal tissues can capture HIV-1 and migrate to lymphoid tissues, where they become mature DCs (mDCs) for effective antigen presentation. DC maturation promotes HIV-1 transmission; however, the underlying mechanisms remain unclear. Here we have compared monocyte-derived iDCs and mDCs for their efficiencies and mechanisms of HIV-1 transmission. We have found that mDCs significantly facilitate HIV-1 endocytosis and efficiently concentrate HIV-1 at virological synapses, which contributes to mDC-enhanced viral transmission, at least in part. mDCs were more efficient than iDCs in transferring HIV-1 to various types of target cells independently of C-type lectins, which partially accounted for iDC-mediated HIV-1 transmission. Efficient HIV-1 trans-infection mediated by iDCs and mDCs required contact between DCs and target cells. Moreover, rapid HIV-1 degradation occurred in both iDCs and mDCs, which correlated with the lack of HIV-1 retention-mediated long-term viral transmission. Our results provide new insights into the mechanisms underlying DC-mediated HIV-1 transmission, suggesting that HIV-1 exploits mDCs to facilitate its dissemination within lymphoid tissues.     

Shaping phenotype, function, and survival of dendritic cells by cytomegalovirus-encoded IL-10

Human dendritic cells (DCs) are essential for the antiviral immune response and represent a strategically important target for immune evasion of viruses, including human CMV (HCMV). Recently, HCMV has been discovered to encode a unique IL-10 homologue (cmvIL-10). In this study we investigated the capacity of cmvIL-10 to shape phenotype, function, and survival of DCs. For comparison we included human IL-10 and another IL-10 homologue encoded by EBV, which does not directly target DCs. Interestingly, cmvIL-10 strongly activated STAT3 in immature DCs despite its low sequence identity with human IL-10. For most molecules cmvIL-10 blocked LPS-induced surface up-regulation, confirming its role as an inhibitor of maturation. However, a small number of molecules on LPS-treated DCs including IDO, a proposed tolerogenic molecule, showed a different behavior and were up-regulated in response to cmvIL-10. Intriguingly, the expression of C-type lectin DC-specific ICAM-grabbing nonintegrin, a receptor for HCMV infection found exclusively on DCs, was also enhanced by cmvIL-10. This phenotypic change was mirrored by the efficiency of HCMV infection. Moreover, DCs stimulated with LPS and simultaneously treated with cmvIL-10 retained the function of immature DCs. Finally, cmvIL-10 increased apoptosis associated with DC maturation by blocking up-regulation of the antiapoptotic long form cellular FLIP. Taken together, these findings show potential mechanisms by which cmvIL-10 could assist HCMV to infect DCs and to impair DC function and survival.     

Human peripheral blood monocyte-derived interleukin-10-induced semi-mature dendritic cells induce anergic CD4(+) and CD8(+) T cells via presentation of the internalized soluble antigen and cross-presentation of the phagocytosed necrotic cellular fragments

We examined the ability of human monocyte-derived interleukin (IL)-10-induced semi-mature dendritic cells (semi-mDCs) that had been pulsed with soluble protein and necrotic cellular fragments to induce an antigen (Ag)-specific anergy in CD4(+) and CD8(+) T cells. IL-10 converted normal immature DCs (iDCs) into semi-mDCs during the maturation. In contrast to normal iDCs and mature DCs, IL-10-induced semi-mDCs as well as IL-10-treated iDCs not only had reduced their allogeneic T cell-stimulatory capacity, but also induced an allogeneic Ag-specific anergy in T cells. Normal mDCs that had been pulsed with tetanus toxin (TT) or allogeneic necrotic cellular fragments caused further activation of TT-specific CD4(+) T cells or allogeneic fibroblast-specific CD8(+) T cells, Ag-pulsed IL-10-induced semi-mDCs induced an anergic state in both cell types. Thus, our results suggest that IL-10-induced semi-mDCs induce an Ag-specific anergy in CD4(+) and CD8(+) T cells via presentation of the internalized protein and cross-presentation of the phagocytosed cellular fragments.     

MicroRNA-146a and MicroRNA-146b Regulate Human Dendritic Cell Apoptosis and Cytokine Production by Targeting TRAF6 and IRAK1 Proteins

We have previously reported 27 differentially expressed microRNAs (miRNAs) during human monocyte differentiation into immature dendritic cells (imDCs) and mature DCs (mDCs). However, their roles in DC differentiation and function remain largely elusive. Here, we report that microRNA (miR)-146a and miR-146b modulate DC apoptosis and cytokine production. Expression of miR-146a and miR-146b was significantly increased upon monocyte differentiation into imDCs and mDCs. Silencing of miR-146a and/or miR-146b in imDCs and mDCs significantly prevented DC apoptosis, whereas overexpressing miR-146a and/or miR-146b increased DC apoptosis. miR-146a and miR-146b expression in imDCs and mDCs was inversely correlated with TRAF6 and IRAK1 expression. Furthermore, siRNA silencing of TRAF6 and/or IRAK1 in imDCs and mDCs enhanced DC apoptosis. By contrast, lentivirus overexpression of TRAF6 and/or IRAK1 promoted DC survival. Moreover, silencing of miR-146a and miR-146b expression had little effect on DC maturation but enhanced IL-12p70, IL-6, and TNF-α production as well as IFN-γ production by IL-12p70-mediated activation of natural killer cells, whereas miR-146a and miR-146b overexpression in mDCs reduced cytokine production. Silencing of miR-146a and miR-146b in DCs also down-regulated NF-κB inhibitor IκBα and increased Bcl-2 expression. Our results identify a new negative feedback mechanism involving the miR-146a/b-TRAF6/IRAK1-NF-κB axis in promoting DC apoptosis.     

Human immunodeficiency virus type 1 gp120 induces abnormal maturation and functional alterations of dendritic cells: a novel mechanism for AIDS pathogenesis

Dendritic cells (DCs) play a crucial role in bridging innate and acquired immune responses to pathogens. In human immunodeficiency virus type 1 (HIV-1) infection, immature DCs (iDCs) are also main targets for HIV-1 at the mucosal level. In this study, we evaluated the effects of HIV-1-DC interactions on the maturation and functional activity of these cells. Exposure of human monocyte-derived iDCs to either aldrithiol-2-inactivated HIV-1 or gp120 led to an upmodulation of activation markers indicative of functional maturation. Despite their phenotype, these cells retained antigen uptake capacity and showed an impaired ability to secrete cytokines or chemokines and to induce T-cell proliferation. Although gp120 did not interfere with DC differentiation, the capacity of these cells to produce interleukin-12 (IL-12) upon maturation was markedly reduced. Likewise, iDCs stimulated by classical maturation factors in the presence of gp120 lacked allostimulatory capacity and did not produce IL-12, in spite of their phenotype typical of activated DCs. Exogenous addition of IL-12 restores the allostimulatory capacity of gp120-exposed DCs. The finding that gp120 induces abnormal maturation of DCs linked to profound suppression of their activities unravels a novel mechanism by which HIV can lead to immune dysfunction in AIDS patients.     

Inhibition of myeloid dendritic cell accessory cell function and induction of T cell anergy by alcohol correlates with decreased IL-12 production

Alcohol consumption inhibits accessory cell function and Ag-specific T cell responses. Myeloid dendritic cells (DCs) coordinate innate immune responses and T cell activation. In this report, we found that in vivo moderate alcohol intake (0.8 g/kg of body weight) in normal volunteers inhibited DC allostimulatory capacity. Furthermore, in vitro alcohol treatment during DC differentiation significantly reduced allostimulatory activity in a MLR using naive CD4(+) T cells, and inhibited tetanus toxoid Ag presentation by DCs. Alcohol-treated DCs showed reduced IL-12, increased IL-10 production, and a decrease in expression of the costimulatory molecules CD80 and CD86. Addition of exogenous IL-12 and IL-2, but not neutralization of IL-10, during MLR ameliorated the reduced allostimulatory capacity of alcohol-treated DCs. Naive CD4(+) T cells primed with alcohol-treated DCs showed decreased IFN-gamma production that was restored by exogenous IL-12, indicating inhibition of Th1 responses. Furthermore, CD4(+) T cells primed with alcohol-treated DCs were hyporesponsive to subsequent stimulation with the same donor-derived normal DCs, suggesting the ability of alcohol-treated DCs to induce T cell anergy. LPS-induced maturation of alcohol-treated immature DCs partially restored the reduced allostimulatory activity, whereas alcohol given only during DC maturation failed to inhibit DC functions, suggesting that alcohol primarily impairs DC differentiation rather than maturation. NFkappaB activation, a marker of DC maturation was not affected by alcohol. Taken together, alcohol both in vitro and in vivo can impair generation of Th1 immune responses via inhibition of DC differentiation and accessory cell function through mechanisms that involve decreased IL-12 induction.     

The effect of LIGHT in inducing maturation of monocyte-derived dendritic cells from MDS patients

LIGHT is a recently cloned novel cytokine belonging to the TNF family that is selectively expressed on immature dendritic cells (iDCs) generated from monocytes isolated from human PBMCs. In these studies, we demonstrate that exogenous soluble LIGHT or soluble CD40 ligand (CD40L) can promote monocyte-derived dendritic cell maturation in vitro by the up-regulation of CD86, CD80, CD83, and HLA-DR antigen expression. Immature dendritic cells differentiated from monocytes of MDS patients displayed lower levels of costimulatory and HLA-DR molecules compared with iDCs differentiated from monocytes of normal subjects. However, upon induction of maturation by LIGHT or CD40L, the expression of costimulatory and HLA-DR molecules is comparable between DCs from MDS and normal subjects. Exogenous LIGHT- and CD40L-stimulated mature DCs (mDCs) also displayed increased antigen presentation to autologous T lymphocytes (tetanus toxin) or allogeneic T lymphocytes in mixed lymphocyte reactions. DCs matured by LIGHT showed increased secretion of IL-6, IL-12p75, and TNF-, but not IL-1. We conclude that both LIGHT and CD40L are immunoregulating factors that induce monocyte-derived iDCs from MDS patients to undergo maturation resulting in increased antigen presentation and T-cell activation. Monocyte-derived DCs can be stimulated to undergo phenotypic and functional changes with LIGHT that might be applied in the development of a DC-based vaccine for MDS treatment.     

The immunostimulatory effects of retinoblastoma cell supernatant on dendritic cells

Dendritic cells (DCs) are crucial for the induction and maintenance of tumor-specific immune responses. Studies have shown that tumor-associated DCs are immunosuppressed in some human tumors. However, phenotype and function of DCs in retinoblastoma (RB) remain unclear. RB cell supernatant (RBcs) was used to treat DCs in vitro to explore the effect of RB cells on DCs. DCs were generated from peripheral blood mononuclear cells of healthy donors. On day 5 of culture, DCs were treated with RBcs for 24 h, and then purified using magnetic beads. The maturation of DCs was induced by TNF-α or LPS. After treatment with RBcs, expression of co-stimulatory molecules CD80 and CD86 was elevated in DCs, accompanied by increased production of IL-12p70, TNF-α, IL-6, IL-1β, and IL-8 but decreased production of IL-10. RBcs neither inhibited DC maturation nor promoted DC apoptosis. Moreover, RBcs-exposed DCs stimulated allogenetic T cell proliferation and T cell-derived cytokine production. These results indicate that RBcs can improve DCs’ antigen presenting function and capability to activate T cells, suggesting that RB cells may have an immunostimulatory effect on DCs, and Dcbased immunotherapy may be adopted in the treatment of RB.     

VEGF-A-induced immature DCs not mature DCs differentiation into endothelial-like cells through ERK1/2-dependent pathway

Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in anti-tumour immunity. Endothelial-like differentiation of DCs is an interesting phenomenon. The specific role of vascular endothelial growth factor-A (VEGF-A) on the differentiation of immature DCs (iDCs) and mature DCs (mDCs) is worth further research. Here, we show that VEGF-A can induce iDCs to differentiate into endothelial-like cells (ELCs). But it has no obvious influence on mDCs. In the process of endothelial-like differentiation of iDCs, a sustained activation of extracellular signal-regulated kinase (ERK1/2) and cAMP response element binding protein (CREB) was detected. VEGF-A induced the activation of ERK1/2, and led to the nuclear translocation of phosphorylation ERK1/2. Incubation of iDCs with the ERK1/2 upstream kinase MEK1/2 inhibitor PD98059, blocked the phosphorylation of ERK1/2 and CREB as well as the endothelial-like differentiation of iDCs. These data suggest that VEGF-A induces endothelial-like differentiation of iDCs not mDCs through ERK1/2 signalling pathway.     

Infection of dendritic cells by a gamma2-herpesvirus induces functional modulation

The murine gamma-herpesvirus-68 (gammaHV68) establishes viral latency in dendritic cells (DCs). In the present study, we examined the specific consequences of DC infection by gammaHV68, both in vivo and in vitro. Ex vivo analysis of infected mice showed that the virus colonizes respiratory DCs very early after infection and that all subsets of splenic DCs analyzed are viral targets. We have developed and characterized an in vitro model of gammaHV68 infection of DCs. Using this model, we demonstrated that viral infection neither induces full DC maturation nor interferes with exogenous activation, which is assessed by cell surface phenotypic changes. However, whereas gammaHV68 infection alone failed to elicit cytokine secretion, IL-10 secretion of exogenously activated DCs was enhanced. Furthermore, gammaHV68-infected DCs efficiently stimulated virus-specific T cell hybridomas but failed to induce alloreactive stimulation of normal T cells. These data indicate that viral infection doesn't interfere with Ag processing and presentation but does interfere with the ability of DCs to activate T cells. The inhibition of T cell activation was partially reversed by blocking IL-10. Analysis of infected mice shows elevated levels of IL-10 expression in DCs and that lack of endogenous IL-10 is associated with decreased gammaHV68 long-term latency. Taken together, these observations indicate that gamma2-herpesvirus infection of DCs is a mechanism of viral immune evasion, partially mediated by IL-10.     

Selective regulation of interleukin-10 production via Janus kinase pathway in murine conventional dendritic cells

In this study, we examined the role of JAKs in regulation of inflammatory versus anti-inflammatory cytokine balance in murine conventional dendritic cells (DCs). Highly purified lipopolysaccharide (upLPS) combined with imiquimod (IQ) synergistically induced IL-10 production by DCs, while each ligand alone showed a slight effect on the IL-10 production. Marked phosphorylation of JAK2, STAT1 and STAT3 was detected in DCs following upLPS plus IQ stimulation. Blocking the JAK pathway by JAK inhibitor I (JAKi) resulted in significant inhibition of IL-10 production by the DCs. However, JAKi showed negligible effect on the DC production of IL-12, IL-6 and TNF-α. JAKi completely blocked the TLR-mediated STATs activation, and attenuated the activation of Akt, a downstream effector of PI3K, in DCs stimulated by upLPS plus IQ. LY294002, a specific inhibitor of PI3K, markedly inhibited the DC production of IL-10. Thus, JAK-PI3K axis appeared to be responsible for the IL-10 production by DCs.