12/15-Lipoxygenase-mediated enzymatic lipid oxidation regulates DC maturation and function.

DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell-driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase-meditated (12/15-LO-mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO-encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO-deficient DCs to 12/15-LO-derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO-deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune encephalomyelitis in mice lacking 12/15-LO resulted in an exacerbated Th17-driven autoimmune disease. Together, our data reveal that 12/15-LO controls maturation of DCs and implicate enzymatic lipid oxidation in shaping the adaptive immune response.

Topical application of soluble CD83 induces IDO-mediated immune modulation, increases Foxp3+ T cells, and prolongs allogeneic corneal graft survival.

Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-ß. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-ß, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.

The IL-2 diphtheria toxin fusion protein denileukin diftitox modulates the onset of diabetes in female nonobese diabetic animals in a time-dependent manner and breaks tolerance in male nonobese diabetic animals.

Denileukin diftitox, also known as DAB(389)IL-2 or Ontak, is a fusion protein toxin consisting of the full-length sequence of the IL-2 protein and as toxophore the truncated diphtheria toxin. As a consequence, it delivers the toxic agent to CD25-bearing cells, whereby CD25 represents the high-affinity α-subunit of the IL-2 receptor. Initially it was developed for the treatment of patients with cutaneous T cell lymphoma. Meanwhile, denileukin diftitox is also used as an adjuvant in other tumor therapies and neoplastic disorders. In this study, to our knowledge we report for the first time that denileukin diftitox has also dramatic effects regarding the pathology of type 1 diabetes using the NOD mouse model. Repeated injections of denileukin diftitox into female NOD mice at 12 wk of age led to a clear acceleration of disease onset, whereas injection at 7 wk of age did not. Using male NOD mice, which are much less susceptible to diabetes, we demonstrate that the injection of denileukin diftitox leads to a dramatic development of type 1 diabetes within days after injection, thereby obviously breaking pre-existing tolerance mechanisms. This is accompanied by an increased IFN-γ production of autoreactive splenic cells and a decreased presence of regulatory CD4(+)CD25(+)Foxp3(+) T cells. In contrast, transfer of CD4(+)CD25(+)Foxp3(+) T cells could correct the defect after denileukin diftitox treatment. Furthermore, whereas IFN-γ production was increased in the pancreata of treated animals, insulin expression was strongly reduced. These finding should be considered when denileukin diftitox is used for the treatment of patients suffering from tumors and/or autoimmune disorders.

MCS-18, a novel natural plant product prevents autoimmune diabetes.

There is still a vital need for new therapies in order to prevent or treat type I diabetes. In this respect, we report that MCS-18 a novel natural product isolated from the plant Helleborus purpurascens (i.e. Christmas rose) is able to increase diabetes free survival using the NOD-mouse model, which is accompanied with a diminished IFN-γ secretion of splenocytes. In the animal group which has been treated with MCS-18 during week 8 and week 12 of age 70% of the animals showed a diabetes free survival at week 30, whereas in contrast in the untreated animals less than 10% were free of diabetes. MCS-18 treatment significantly reduced islet T-cell infiltrates as well as the rate of T-cell proliferation. Periinsular infiltrates in the MCS-18 treated animals showed a significantly enhanced number of Foxp3(+) CD25(+) T cells, indicating the increased presence of regulatory T cells. These studies show that MCS-18 exerts an efficient immunosuppressive activity with remarkable potential for the therapy of diseases characterized by pathological over-activation of the immune system.

Myeloid-derived suppressor cell activation by combined LPS and IFN-gamma treatment impairs DC development.

Myeloid-derived suppressor cells (MDSC) and DC are major controllers of immune responses against tumors or infections. However, it remains unclear how DC development and MDSC suppressor activity both generated from myeloid precursor cells are regulated. Here, we show that the combined treatment of BM-derived MDSC with LPS plus IFN-gamma inhibited the DC development but enhanced MDSC functions, such as NO release and T-cell suppression. This was not observed by the single treatments in vitro. In the spleens of healthy mice, we identified two Gr-1(low)CD11b(high)Ly-6C(high)SSC(low)Mo-MDSC and Gr-1(high)CD11b(low)PMN-MDSC populations with suppressive potential, whereas Gr-1(high)CD11b(high) neutrophils and Gr-1(low)CD11b(high)SSC(low) eosinophils were not suppressive. Injections of LPS plus IFN-gamma expanded these populations within the spleen but not LN leading to the block of the proliferation of CD8(+) T cells. At the same time, their capacity to develop into DC was impaired. Together, our data suggest that spleens of healthy mice contain two subsets of MDSC with suppressive potential. A two-signal-program through combined LPS and IFN-gamma treatment expands and fully activates MDSC in vitro and in vivo.

Inhibition of the proteasome influences murine and human dendritic cell development in vitro and in vivo.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) known today and are designated as nature's adjuvant since they are the only antigen-presenting cell type capable of inducing naïve T cell responses in vivo. In order to become potent T cell stimulators DC have to mature. This mature DC phenotype is characterized amongst other characteristics by the up-regulation of co-stimulatory molecules such as CD40, CD80, CD86 and the cell surface expression of CD83. Inhibition of their expression blocks the immune responses in vitro and in vivo, and thus represents an interesting strategy to control undesired and/or over-activated immune responses such as in autoimmune disorders, transplant rejections and allergies. Here we investigated the in vitro and in vivo effects of the proteasome inhibitor Velcade in respect to DC phenotype and DC functions in murine and human DC. Interestingly, in vitro, DC maturation as well as DC-mediated T cell stimulation and cytokine production was impaired. Furthermore, administration of the inhibitor in vivo resulted in a reduced mature phenotype of ex vivo generated murine DC. Thus, inhibition of the proteasome interferes with DC maturation and subsequently with DC-mediated T cell stimulation events.

Modulation of murine bone marrow-derived dendritic cells and B-cells by MCS-18 a natural product isolated from Helleborus purpurascens.

MCS-18, a natural product isolated from Helleborus purpurascens has been shown to have several beneficial effects in inflammatory and autoimmune disorders. However, very little is known regarding the immuno-modulatory capacity of MCS-18 in respect to murine bone marrow-derived dendritic cells (BM-DC) and B-cells. Thus, in the present study we examined the effect of MCS-18 on murine BM-DC and B-cells. Interestingly MCS-18 inhibited the expression of important DC-specific molecules and lead to an impaired T-cell stimulation capacity. In addition, MCS-18 also reduced B-cell proliferation and immunoglobulin production.

TLR9 cooperates with TLR4 to increase IL-12 release by murine dendritic cells.

Toll-like receptors (TLR) are expressed on the surface or intracellularly by dendritic cells (DC) and recognize specifically different pathogen-associated molecular patterns (PAMPs). Increasing evidence suggests that TLR expressed by DC can cooperate to synergize their functions. Here, we describe the cooperation of TLR9 and TLR4 triggering of murine bone marrow derived DC by CpG oligonucleotides and LPS, respectively. The simultaneous DC stimulation of LPS and CpG showed additive effects on the production of IL-12 but not on other cytokines, such as TNF, IL-6 or IL-10. CpG pretreatment before LPS induced five times more IL-12p40 and IL-12p70 production by DC, whereas LPS pretreatment before CpG showed no effect. The optimal time interval between CpG and LPS treatment was 4h and the synergistic effects were dependent on myeloid differentiation factor 88 (MyD88) but independent from the DNA backbone and did not mediate by nucleosome remodeling. The stimulatory effect could be further enhanced by addition of IFN-gamma but not anti-CD40 antibodies. These data show, that TLR4 and TLR9 can cooperate to increase selectively IL-12 production by DC.

Factors influencing the generation of murine dendritic cells from bone marrow: the special role of fetal calf serum.

Since the generation of dendritic cells (DC) from myeloid precursor cells within the bone marrow (BM) was first reported, it has developed to one of the standard tools for studying DC biology in rodent systems. However, antigen-specific effects of such DC on polyclonal T cell populations may be influenced by the additional presentation of bystander antigens derived from fetal calf sera (FCS). This may lead to misinterpretations of the antigen-specific effects on the T cell response. In addition, many factors, including FCS, also influence the quantity and quality of BM-DC already during culture. Here, we review the factors that have an impact on the generation of BM-DC, including criteria regarding the mice, but also technical parameters and the different growth factors. The critical role of FCS in BM-DC cultures and alternative methods using murine sera or serum-free media are discussed.

Dendritic cells matured with TNF can be further activated in vitro and after subcutaneous injection in vivo which converts their tolerogenicity into immunogenicity.

Dendritic cell (DC) maturation can occur by different types of stimuli. Previously, we described that murine DC matured with tumor necrosis factor (TNF) up-regulate surface MHC and costimulatory molecules but lack cytokine release, and therefore termed them semi-mature DC. These TNF/DC-induced tolerance after intravenous (i.v.) injection in a model of experimental autoimmune encephalomyelitis (EAE). Here, we show that TNF/DC are not terminally differentiated but can still respond to the microbial stimulus lipopolysaccharide. Subcutaneously injected TNF/DC induce an unpolarized T(H)1/T(H)2 response and are not protective in the experimental autoimmune encephalomyelitis model. Although TNF/DC home to the draining lymph node, they remain negative for intracellular cytokine stainings. However, the nonmigrating, endogenous DC started to produce interleukin (IL)-12p40, TNF and little IL-6, IL-10, and MCP-1 in a bystander fashion. Together, DC matured with the inflammatory stimulus TNF remains responsive to further signals in vitro and in vivo. These signals can be provided by pathogens or the subcutaneous injection route, which can convert them from tolerogenic to immunogenic DC. These findings are important for selecting the appropriate injection route of human DC for tumor immunotherapy.

Myeloid dendritic cell precursors generated from bone marrow suppress T cell responses via cell contact and nitric oxide production in vitro.

Tolerogenic activity of myeloid dendritic cells (DC) has so far been attributed mostly to immature or semi-mature differentiation stages but never to their precursor cells. Although myeloid suppressor cells (MSC) have been isolated ex vivo, their developmental relationship to DC and their precise phenotype remained elusive. Here, we describe the generation of MSC as myeloid DC precursors with potent suppressive activity on allogeneic and OVA-specific CD4+ and CD8+ T cell responses in vitro. These MSC appear transiently in DC cultures of bone marrow (BM) cells after 8-10 days under low GM-CSF conditions or after 3-4 days under high GM-CSF conditions. They represent CD11c- myeloid precursor cells with ring-shaped nuclei and are Gr-1low (i.e. Ly-6C+, Ly-6Glow), CD11b+, CD31+, ER-MP58+, asialoGM1+ and F4/80+. Sorted MSC develop into CD11c+ DC within 6 days. Their suppressor activity partially depends on IFN-gamma stimulation. Suppression is mediated through mechanisms requiring cell contact and nitric oxide but is independent of TNF, CD1d and TGF-beta. Together, our data describe the generation of MSC with distinct suppressor mechanisms in vitro preceding their development into immature DC.

IL-4 supports the generation of a dendritic cell subset from murine bone marrow with altered endocytosis capacity.

Dendritic cells (DC) of myeloid origin can be generated from mouse bone marrow (BM) using granulocyte macrophage-colony stimulating factor (GM-CSF). Immature major histocompatibility complex (MHC) II(low) DC are known to bear a high endocytosis capacity, in contrast to DC precursors and mature DC. Now we found that a subset of MHC II(low) DC in BM-DC cultures is unable to exert mannose receptor-mediated endocytosis of fluorescein isothiocyanate (FITC)-dextran (DX) and resembles immature Langerhans cells (LC). The FITC-DX endocytosis activity of LC-like cells occurs at an earlier stage of development, where the surface MHC II expression is absent or very weak. This LC-like subset expresses higher levels of E-cadherin but lower amounts of the markers Gr-1, scavenger receptor 2F8, and CD11b, when compared with the highly endocytic DC subset. The latter myeloid DC resemble monocyte-derived DC (MoDC). The sorted LC-like population develops completely and exclusively into mature MHC IIhigh DC, and the MoDC-like cells remain immature MHC II(low) DC or develop into adherent MHC IIneg macrophages or mature into MHC IIhigh DC. The development of LC-like cells is promoted by interleukin-4. Thus, we show here that the simultaneous development of LC-like and MoDC-like DC subsets occurs in standard bulk cultures with GM-CSF, suggesting the existence of two different precursors for LC and MoDC in BM.

Interleukin-3Ralpha+ myeloid dendritic cells and mast cells develop simultaneously from different bone marrow precursors in cultures with interleukin-3.

The distinct developmental routes of dendritic cells and mast cells from murine bone marrow cultures with interleukin-3 are unclear. We found that short-term bone marrow cultures with interleukin-3 after 8-10 d consist of about 10%-30% dendritic cells and 70%-90% mast cell precursors, and only after 4-6 wk do homogeneous populations of mast cells emerge. Phenotypical and functional analysis of interleukin-3/dendritic cells revealed a high similarity with myeloid dendritic cells generated with granulocyte-macrophage colony stimulating factor in the expression of myeloid dendritic cell markers (CD11c+ B220- CD8alpha- CD11b+), major histocompatibility complex II and costimulatory molecules, endocytosis, maturation potential, interleukin-12 production, and T cell priming. Interleukin-3/dendritic cells expressed higher levels of interleukin-3 receptor, however. To dissect the interleukin-3/dendritic cell and mast cell development, we sorted fresh bone marrow cells into six subsets by the antibodies ER-MP12 (CD31) and ER-MP20 (Ly-6C). Both interelukin-3/dendritic cells and granulocyte-macrophage colony stimulating factor/dendritic cells develop from the same bone marrow populations, including the ER-MP12neg, ER-MP20high bone marrow monocytes. In contrast, mast cells only developed from ER-MP12(int+high), ER-MP20neg bone marrow cell subsets, indicating that different precursors exist for interleukin-3/dendritic cells and mast cells. Established mast cell cultures could not be converted to dendritic cells or stimulated to express major histocompatibility complex II molecules in vitro or home to lymph node T cell areas in vivo. In summary, we show that dendritic cells generated from bone marrow precursors with interleukin-3 are clearly myeloid and develop via a different pathway compared to bone marrow mast cells.

Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF.

Little is known about the distinct roles of the two types of IL-4R on DC. Here we report that IL-4 and IL-13 are able to promote DC maturation, as evaluated by up-regulation of MHC class II and costimulatory molecules, when the concentration of GM-CSF is relatively lower than the dose of IL-4 or IL-13. In addition, under these conditions both cytokines enable DC to respond to maturation stimuli such as bacterial products or proinflammatory cytokines. Both IL-4 and IL-13 act synergistically with weak maturation stimuli such as TNF-alpha or CD40. The IL-4R signaling for DC maturation requires the IL-4R alpha-chain and STAT6, but not Janus kinase 3, indicating that IL-4R type II signaling is preferentially responsible for these effects. In contrast, the production of IL-12 p70, but not IL-10 and TNF, induced by microbial products was enhanced only by IL-4, not by IL-13 or Y119D, a selective type II IL-4R agonist, in vitro and in vivo. This enhancement was dependent on the presence of Janus kinase 3, indicating that this function is exclusively mediated by the type I IL-4R. In short, we discerned the individual roles of the two IL-4R types on DC function, showing that IL-4R type I promotes IL-12 secretion independently of GM-CSF concentration, while IL-4R type II promotes the up-regulation of MHC class II and costimulatory surface markers in a GM-CSF concentration-dependent manner.

Repetitive injections of dendritic cells matured with tumor necrosis factor alpha induce antigen-specific protection of mice from autoimmunity.

Mature dendritic cells (DCs) are believed to induce T cell immunity, whereas immature DCs induce T cell tolerance. Here we describe that injections of DCs matured with tumor necrosis factor (TNF)-alpha (TNF/DCs) induce antigen-specific protection from experimental autoimmune encephalomyelitis (EAE) in mice. Maturation by TNF-alpha induced high levels of major histocompatibility complex class II and costimulatory molecules on DCs, but they remained weak producers of proinflammatory cytokines. One injection of such TNF/DCs pulsed with auto-antigenic peptide ameliorated the disease score of EAE. This could not be observed with immature DCs or DCs matured with lipopolysaccharide (LPS) plus anti-CD40. Three consecutive injections of peptide-pulsed TNF/DCs derived from wild-type led to the induction of peptide-specific predominantly interleukin (IL)-10-producing CD4(+) T cells and complete protection from EAE. Blocking of IL-10 in vivo could only partially restore the susceptibility to EAE, suggesting an important but not exclusive role of IL-10 for EAE prevention. Notably, the protection was peptide specific, as TNF/DCs pulsed with unrelated peptide could not prevent EAE. In conclusion, this study describes that stimulation by TNF-alpha results in incompletely matured DCs (semi-mature DCs) which induce peptide-specific IL-10-producing T cells in vivo and prevent EAE.