1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation

1 Alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D3, is a potent immunomodulatory agent. Here we show that dendritic cells (DCs) are major targets of 1,25(OH)2D3-induced immunosuppressive activity. 1,25(OH)2D3 prevents the differentiation in immature DCs of human monocytes cultured with GM-CSF and IL-4. Addition of 1,25(OH)2D3 during LPS-induced maturation maintains the immature DC phenotype characterized by high mannose receptor and low CD83 expression and markedly inhibits up-regulation of the costimulatory molecules CD40, CD80, and CD86 and of class II MHC molecules. This is associated with a reduced capacity of DCs to activate alloreactive T cells, as determined by decreased proliferation and IFN-gamma secretion in mixed leukocyte cultures. 1, 25(OH)2D3 also affects maturing DCs, leading to inhibition of IL-12p75 and enhanced IL-10 secretion upon activation by CD40 ligation. In addition, 1,25(OH)2D3 promotes the spontaneous apoptosis of mature DCs. The modulation of phenotype and function of DCs matured in the presence of 1,25(OH)2D3 induces cocultured alloreactive CD4+ cells to secrete less IFN-gamma upon restimulation, up-regulate CD152, and down-regulate CD154 molecules. The inhibition of DC differentiation and maturation as well as modulation of their activation and survival leading to T cell hyporesponsiveness may explain the immunosuppressive activity of 1, 25(OH)2D3.     

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 TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.     

Dendritic cell differentiation state and their interaction with NKT cells determine Th1/Th2 differentiation in the murine model of Leishmania major infection

Recent reports demonstrated that dendritic cells (DC) sense inflammatory and microbial signals differently, redefining their classical subdivision into an immature endocytic and a mature Ag-presenting differentiation stage. Although both signals induce DC maturation by up-regulating MHC class II and costimulatory molecules, only TLR signals such as LPS are able to trigger proinflammatory cytokine secretion by DCs, including Th1-polarizing IL-12. Here, we explored the murine Leishmania major infection model to examine the CD4(+) T cell response induced by differentially matured DCs. When partially matured TNF-DCs were injected into BALB/c mice before infection, the mice failed to control L. major infection and developed a Th2 response which was dependent on IL-4Ralpha signaling. In contrast, injections of fully matured LPS+CD40-DCs induced a Th1 response controlling the infection. Pulsing DCs with a lysate of L. major did not affect DC maturation with TNF-alpha or LPS+anti-CD40. When the expression of different Notch ligands on DCs was analyzed, we found increased expression of Th2-promoting Jagged2 in TNF-DCs, whereas LPS+CD40-DCs up-regulated the Th1-inducing Delta4 and Jagged1 molecules. The Th2 polarization induced by TNF-DCs required interaction with CD1d-restricted NKT cells. However, NKT cell activation by L. major lysate-pulsed DCs was not affected by blockade of the endogenous glycolipid, suggesting exchange with exogenous parasite-derived CD1 glycolipid Ag. In sum, the differentiation stage of DCs as well as their interaction with NKT cells determines Th1/Th2 differentiation. These results have generic implications for the understanding of DC-driven Th cell responses and the development of improved DC vaccines against leishmaniasis.     

Galectin-9 induces maturation of human monocyte-derived dendritic cells

Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.     

Cigarette smoke extract suppresses human dendritic cell function leading to preferential induction of Th-2 priming

Dendritic cells (DC) are key regulators of immune responses. In the current study, we hypothesized that cigarette smoke-induced aberrance in DC function is an important mechanism by which smokers develop cancer, infection, and allergy--diseases common in smokers. We demonstrate that cigarette smoke extract (CSE) inhibits DC-mediated priming of T cells, specifically inhibiting the secretion of IFN-gamma whereas enhancing the production of IL-4 in the MLR. Conditioning with CSE did not effect cytokine (IL-10, IL-6, or IL-12) production from immature DCs, but significantly inhibited IL-12p70 release by LPS-matured DCs. In contrast, IL-10 secretion by LPS-activated CSE-conditioned DCs was enhanced when compared with control DCs. CSE also induced cyclooxygenase-2 protein levels in maturing DCs and significantly augmented endogenous PGE2 release. Conditioning of DCs with CSE also suppressed LPS-mediated induction of CD40, CD80, and CD86, and suppressed maturation-associated CCR7 expression. Although CSE has been reported to induce apoptosis of fibroblasts and epithelial cells, the immunomodulatory effects observed with CSE were not due to diminished DC viability. The effects of CSE on DC function were not exclusively mediated by nicotine, because equivalent, or even higher concentrations of nicotine than those found in CSE, failed to suppress DC-induced T cell priming. These data provide evidence that soluble components extracted from cigarette smoke suppress key DC functions and favor the development of Th-2 immunity.     

IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation

Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130(F759/F759)) or STAT3 (gp130(FxxQ/FxxQ)), and combined gp130 and IL-6 defects (gp130(F759/F759)/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130(F759/F759) mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.     

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.     

The endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces dendritic cell maturation

Angiogenesis is an essential component of chronic inflammation that is linked to carcinogenesis. In this study, we report that human vascular endothelial growth inhibitor (VEGI, TNF superfamily 15), an endothelial cell-produced antiangiogenic cytokine, induces mouse dendritic cell (DC) maturation, a critical event in inflammation-initiated immunity. VEGI-stimulated bone marrow-derived immature DCs display early activation of maturation signaling molecules NF-kappaB, STAT3, p38, and JNK, and cytoskeleton reorganization and dendrite formation. The activation signals are partially inhibited by using a neutralizing Ab against death domain-containing receptor-3 (DR3) or a truncated form of DR3 consisting of the extracellular domain, indicating an involvement of DR3 in the transmission of VEGI activity. A VEGI isoform, TL1A, does not induce similar activities under otherwise identical experimental conditions. Additionally, the cells reveal significantly enhanced expression of mature DC-specific marker CD83, secondary lymphoid tissue-directing chemokine receptor CCR7, the MHC class-II protein (MHC-II), and costimulatory molecules CD40, CD80, and CD86. Functionally, the cells exhibit decreased Ag endocytosis, increased cell surface distribution of MHC-II, and increased secretion of IL-12 and TNF. Moreover, VEGI-stimulated DCs are able to facilitate the differentiation of CD4+ naive T cells in cocultures. These findings suggest that the anticancer activity of VEGI arises from coupling the inhibition of endothelial cell growth with the promotion of the adaptive immune mechanisms through the stimulation of DC maturation.     

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-α in lung cancer

INTRODUCTION: Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. RESULTS: Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. CONCLUSION: These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.     

Morphine reciprocally regulates IL-10 and IL-12 production by monocyte-derived human dendritic cells and enhances T cell activation

We evaluated the effect of morphine on human dendritic cells (DCs). Interestingly, immature DCs were found to express all 3 (mu, kappa, delta) opioid receptors on the cell surface. Chronic morphine treatment (10(-8) to 10(-12) M) during the development of DCs from monocytes augmented LPS-induced upregulation of HLA-DR, CD86, CD80, and CD83 and increased the T cell stimulatory capacity of DCs, which could be inhibited by naloxone, an opioid receptor antagonist. The change in surface phenotype was paralleled by a p38 MAPK-dependent decrease in IL-10 and increase in IL-12 secretion. Our data indicate that morphine exerts an immunostimulatory effect by modulating LPS-induced DC maturation.     

Developmental Regulation by Cytokines of Bone Marrow-Derived Dendritic Cells and Epidermal Langerhans Cells

Dendritic cells (DC) are specialized antigen-presenting cells involved in T cell-mediated immune responses. Differentiation and functional maturation of the DC are now known to be regulated by various cytokines, including TGF-β1. The experiments of this study examined the effect of other cytokines, such as IL-4, IL-10 and IL-6, on the differentiation and maturation of bone marrow (BM)-derived DC (BM-DC) and epidermal Langerhans cells (LC). When IL-6 or IL-10 was added to cultures of BM cells in the presence of GM-CSF, both cytokines, as in the case of TGF-β1, suppressed the maturation of DC in terms of the expression of adhesion and costimulatory molecules and T cell-stimulating activity. In contrast, IL-4 was not suppressive but rather supportive for the differentiation of DC. However, these suppressive cytokines hardly counteracted the maturation-inducing activity of TNF-α when added to cultures of immature DC. In addition, they appeared to block the overmaturation of DC, which is characterized by a loss of MHC class II molecules. Regarding LC maturation in epidermal cell cultures, IL-6 and IL-10 were inhibitory for the expression of CD86 and CD80 in a dose-dependent fashion. Unlike BM-DC, LC maturation was slightly enhanced by TGF-β1. The protein antigen-presentation by LC to Th1 clone was not affected by IL-6, but slightly reduced by IL-10. These results suggest that each cytokine contributes to regulate the differentiation and maturation of DC at a different developmental stage.     

On/off TLR signaling decides proinflammatory or tolerogenic dendritic cell maturation upon CD1d-mediated interaction with invariant NKT cells

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.     

Involvement of LEK1 in dendritic cell regulation of T cell immunity against Chlamydia

We investigated the hypothesis that the enhanced Ag-presenting function of IL-10-deficient dendritic cells (DCs) is related to specific immunoregulatory cytoskeletal molecules expressed when exposed to Ags. We analyzed the role of a prominent cytoskeletal protein, LEK1, in the immunoregulation of DC functions; specifically cytokine secretion, costimulatory molecule expression, and T cell activation against Chlamydia. Targeted knockdown of LEK1 expression using specific antisense oligonucleotides resulted in the rapid maturation of Chlamydia-exposed DCs as measured by FACS analysis of key activation markers (i.e., CD14, CD40, CD54, CD80, CD86, CD197, CD205, and MHC class II). The secretion of mostly Th1 cytokines and chemokines (IL-1a, IL-9, IL-12, MIP-1a, and GM-CSF but not IL-4 and IL-10) was also enhanced by blocking of LEK1. The function of LEK1 in DC regulation involves cytoskeletal changes, since the dynamics of expression of vimentin and actin, key proteins of the cellular cytoskeleton, were altered after exposure of LEK1 knockdown DCs to Chlamydia. Furthermore, targeted inhibition of LEK1 expression resulted in the enhancement of the immunostimulatory capacity of DCs for T cell activation against Chlamydia. Thus, LEK1 knockdown DCs activated immune T cells at least 10-fold over untreated DCs. These results suggest that the effect of IL-10 deficiency is mediated through LEK1-related events that lead to rapid maturation of DCs and acquisition of the capacity to activate an elevated T cell response. Targeted modulation of LEK1 expression provides a novel strategy for augmenting the immunostimulatory function of DCs for inducing an effective immunity against pathogens.     

Dendritic cell maturation occurs through the inhibition of GSK-3β

Dendritic cell (DC) maturation results in changes in antigen processing and presentation, governing the fate of adaptive immunity. Understanding the intracellular signaling pathways governing DC maturation is therefore critical. In this study, we observed that the kinase, GSK-3β, is present in its active form in resting immature DCs isolated from the spleen and bone marrow of mice. Induction of DC maturation using GM-CSF, IL-4 and TNF-α resulted in GSK-3β inhibition, as reflected by increased phosphorylation of Serine 9 on the kinase, and concomitant stabilization of its substrate, β-catenin. Treatment of immature DCs with a GSK-3β inhibitor increased cell surface expression of CD80, CD86 and CD40 on DCs, enhancing their ability to present antigen and activating IL-2 secretion by T cells. GSK-3β inhibition also parallels dendritic cell maturation in vivo. Our results show that GSK-3β signaling controls DC maturation and suggest that this kinase could be manipulated to modulate adaptive immunity.     

Expressional and functional studies of CKLF1 during dendritic cell maturation

Chemokine-like factor 1 (CKLF1) was the first member of the CKLF-like MARVEL transmembrane domain containing member (CMTM) family to be discovered. Its expression level is increased clearly in peripheral blood lymphocytes upon phytohemagglutinin stimulation, but little is known about the expression and function of CKLF1 in dendritic cells (DCs), which are the most potent antigen-presenting cells. In the present study, we showed that CKLF1 was highly expressed in monocytes. During differentiation from monocytes to immature DCs, CKLF1 was increased dramatically on day 2 and then decreased from day 3 to 5. Upon maturation with different stimuli, CKLF1 was down-regulated. Two peptides of CKLF1, C19 and C27, stimulated the effect of immature DCs (imDCs) on T-cell proliferation and IFN-γ production. Further study on DC maturation showed that C19 and C27 up-regulated HLA-DR expression and IL-12 secretion, with no obvious effects on CD80, CD83 or CD86. Thus, CKLF1-C19 and -C27 can stimulate antigen-presenting capability of imDCs.     

Folate deficiency affects dendritic cell function and subsequent T helper cell differentiation

Insufficient folate status may be related to the increasing prevalence of immune- or inflammation-related chronic diseases. To investigate the effects of folate on immune regulation, we examined the impact of folate deficiency (FD) on dendritic cell (DC) maturation and function and, thus, T helper (Th) cells differentiation. First, bone marrow-derived DCs (BMDCs) were generated from BALB/c mice bone marrow cells cultured in folate-containing (F-BMDCs) or folate-deficient (FD-BMDCs) medium. FD-BMDC displayed more immature phenotype including reduced levels of major histocompatibility complex class II (MHC II), co-stimulatory molecules and characteristic of higher endocytic activity. FD-BMDC produced less IL-12p70 and proinflammatory cytokines in response to lipopolysaccharide. This aberrant DC maturation due to FD resulted in reduced BMDC-induced Th cell activity and lower IL-2, IFNγ, IL-13 and IL-10 productions. Further in vivo study confirmed significantly lower IFNγ and IL-10 productions by T cells and showed higher splenic naïve Th and lower memory T, effector T and regulatory T cell (Treg) percentages in mice fed with the FD diet for 13 weeks. To investigate the role of DCs on T cell activity, splenic DCs (spDC) from FD mice were cocultured with Th cells. The FD spDC had lower MHC II and CD80 expressions and subsequently impaired DC-induced Th differentiation, shown as decreased cytokine productions. This study demonstrated that folate deficiency impaired DC functions and, thus, Th differentiation and responses, suggesting that folate plays a crucial role in maintaining Th cells homeostasis.