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CD83 is a type-I membrane protein and an efficient marker for identifying mature dendritic cells. Whereas membrane-bound, full-length CD83 co-stimulates the immune system, a soluble variant (sCD83), consisting of the extracellular domain only, displays strong immune-suppressive activities. Besides a prediction that sCD83 adopts a V-set Ig-like fold, however, little is known about the molecular architecture of CD83 and the mechanism by which CD83 exerts its function on dendritic cells and additional immune cells. Here, we report the crystal structure of human sCD83 up to a resolution of 1.7 Å solved in three different crystal forms. Interestingly, β-strands C′, C″, and D that are typical for V-set Ig-domains could not be traced in sCD83. Mass spectrometry analyses, limited proteolysis experiments, and bioinformatics studies show that the corresponding segment displays enhanced main-chain accessibility, extraordinary low sequence conservation, and a predicted high disorder propensity. Chimeric proteins with amino acid swaps in this segment show unaltered immune-suppressive activities in a TNF-α assay when compared to wild-type sCD83. This strongly indicates that this segment does not participate in the biological activity of CD83. The crystal structure of CD83 shows the recurrent formation of dimers and trimers in the various crystal forms and reveals strong structural similarities between sCD83 and B7 family members and CD48, a signaling lymphocyte activation molecule family member. This suggests that CD83 exerts its immunological activity by mixed homotypic and heterotypic interactions as typically observed for proteins present in the immunological synapse. [ABSTRACT FROM AUTHOR]

The transmembrane protein CD83, expressed on APCs, B cells, and T cells, can be expressed as a soluble form generated by alternative splice variants and/or by shedding. Soluble CD83 (sCD83) was shown to be involved in negatively regulating the immune response. sCD83 inhibits T cell proliferation in vitro, supports allograft survival in vivo, prevents corneal transplant rejection, and attenuates the progression and severity of autoimmune diseases and experimental colitis. Although sCD83 binds to human PBMCs, the specific molecules that bind sCD83 have not been identified. In this article, we identify myeloid differentiation factor-2 (MD-2), the coreceptor within the TLR4/MD-2 receptor complex, as the high-affinity sCD83 binding partner. TLR4/MD-2 mediates proinflammatory signal delivery following recognition of bacterial LPSs. However, altering TLR4 signaling can attenuate the proinflammatory cascade, leading to LPS tolerance. Our data show that binding of sCD83 to MD-2 alters this signaling cascade by rapidly degrading IL-1R-associated kinase-1, leading to induction of the anti-inflammatory mediators IDO, IL-10, and PGE2 in a COX-2-dependent manner. sCD83 inhibited T cell proliferation, blocked IL-2 secretion, and rendered T cells unresponsive to further downstream differentiation signals mediated by IL-2. Therefore, we propose the tolerogenic mechanism of action of sCD83 to be dependent on initial interaction with APCs, altering early cytokine signal pathways and leading to T cell unresponsiveness. [ABSTRACT FROM AUTHOR]

To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B' core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8(+) T cells. Second, we generated the two-vector-based "modular promoter" system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B' core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. [ABSTRACT FROM AUTHOR]

CD83 is a maturation marker for dendritic cells. In the B cell lineage, CD83 is expressed especially on activated B cells and on light zone B cells during the germinal center (GC) reaction. The function of CD83 during GC responses is unclear. CD83-/- mice have a strong reduction of CD4+ T cells, which makes it difficult to analyze a functional role of CD83 on B cells during GC responses. Therefore, in the present study we generated a B cell-specific CD83 conditional knockout (CD83 B-cKO) model. CD83 B-cKO B cells show defective upregulation of MHC class II and CD86 expression and impaired proliferation after different stimuli. Analyses of GC responses after immunization with various Ags revealed a characteristic shift in dark zone and light zone B cell numbers, with an increase of B cells in the dark zone of CD83 B-cKO mice. This effect was not accompanied by alterations in the level of IgG immune responses or by major differences in affinity maturation. However, an enhanced IgE response was observed in CD83 B-cKO mice. Additionally, we observed a strong competitive disadvantage of CD83-cKO B cells in GC responses in mixed bone marrow chimeras. Furthermore, infection of mice with Borrelia burgdorferi revealed a defect in bacterial clearance of CD83 B-cKO mice with a shift toward a Th2 response, indicated by a strong increase in IgE titers. Taken together, our results show that CD83 is important for B cell activation and modulates GC composition and IgE Ab responses in vivo. [ABSTRACT FROM AUTHOR]

Objective Atherosclerosis is associated with chronic inflammatory responses of the arterial blood vessels. The previously observed protective effect of the MCS-18 substance against the initiation of atherosclerosis in a murine model was explained by its pronounced anti-inflammatory activity. Here, we investigated its impact on murine plaque progression in advanced atherosclerosis and on proatherogenic processes. Approach & Results ApoE-deficient mice were fed a high-fat diet for 12 weeks to induce atherosclerosis, followed by normal chow and intraperitoneal injections of either MCS-18 (500 μg, n = 10) or saline (n = 10) twice a week for another 12 weeks. Plaque size was reduced in MCS-18 treated mice compared to controls (p = 0.001), which was associated with a reduced size of the lipid core (p = 0.01). There was a decrease in apoptotic cells (p = 0.02), endothelial ICAM-1 expression (p < 0.001), and macrophage density (p = 0.01) in the MCS-18 group. In addition, human and murine dendritic cells (DCs) and human umbilical vein endothelial cells (HUVECs) were treated with MCS-18 (50–200 μg/ml) to analyze cell migration and adhesion under flow conditions. MCS-18 reduced human (p = 0.01) and murine (p = 0.006) DC migration. Furthermore, adhesion of MCS-18-treated DCs to a HUVEC monolayer was decreased (p < 0.001). Compared to controls, CD209 (p < 0.001) and CCR7 (p = 0.003) expression was decreased in MCS-18-treated DCs, while in HUVECs lower levels of ICAM-1 (p < 0.001) and of phosphorylated NF-κB-p65 (p = 0.002) were observed. Blocking of ICAM-1 reduced DC adhesion (p < 0.001). Conclusions MCS-18 exhibits interesting therapeutic effects when applied in advanced murine atherosclerosis. Its antiatherogenic impact might be associated with a suppressed adhesion to the endothelium due to down-regulation of endothelial ICAM-1 expression. [ABSTRACT FROM AUTHOR]

Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation. [ABSTRACT FROM AUTHOR]

In the present study we examined the role of thymic stromal lymphopoietin (TSLP) in experimental autoimmune encephalomyelitis (EAE). Here, we report that TSLP knock-out (KO) mice display a delayed onset of disease and an attenuated form of EAE. This delayed onset was accompanied by a reduced number of encephalitogenic T helper type 1 (Th1) cells in the central nervous system (CNS) of TSLP KO mice. In addition, CD4+ and CD8+ T cells from CNS of TSLP KO mice show a reduced activation status in comparison to wild-type mice. It is noteworthy that we could also show that lymph node cells from TSLP KO mice expanded less efficiently and that interleukin (IL)-6-, interferon (IFN)-γ and tumour necrosis factor (TNF)-α levels were reduced. Furthermore, CD3+ T cells isolated in the preclinical phase from myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-immunized TSLP KO mice showed a reduced response after secondary exposure to MOG35-55, indicating that differentiation of naive T cells into MOG35-55-specific effector and memory T cells was impaired in KO mice. The addition of recombinant TSLP enhanced T cell proliferation during MOG35-55 restimulation, showing that T cells also respond directly to TSLP. In summary, these data demonstrate that expression of, and immune activation by, TSLP contributes significantly to the immunopathology of EAE. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The CD83 molecule (CD83) is a well-known surface marker present on mature dendritic cells (mDC). In this study, we show that CD83 is also expressed on a subset of T cells which mediate regulatory T cell (Treg)-like suppressor functions in vitro and in vivo. Treg-associated molecules including CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNFR family-related gene (GITR), Helios and neuropilin-1 (NRP-1) as well as forkhead box protein 3 (FOXP3) were specifically expressed by these CD83+ T cells. In contrast, CD83- T cells showed a naive T cell phenotype with effector T cell properties upon activation. Noteworthy, CD83- T cells were not able to upregulate CD83 despite activation. Furthermore, CD83+ T cells suppressed the proliferation and inflammatory cytokine release of CD83- T cells in vitro. Strikingly, stimulated CD83+ T cells released soluble CD83 (sCD83), which has been reported to possess immunosuppressive properties. In vivo, using the murine transfer colitis model we could show that CD83+ T cells were able to suppress colitis symptoms while CD83- T cells possessed effector functions. In addition, this CD83 expression is also conserved on expanded human Treg. Thus, from these studies we conclude that CD83+ T cells share important features with regulatory T cells, identifying CD83 as a novel lineage marker to discriminate between different T cell populations. [ABSTRACT FROM AUTHOR]

Introduction Inflammation accelerates both plaque progression and instability in the pathogenesis of atherosclerosis. The inhibition of dendritic cell (DC) maturation is a promising approach to suppress excessive inflammatory immune responses and has been shown to be protective in several autoimmune models. The aim of this study was to investigate the immune modulatory effects of the natural substance MCS-18, an inhibitor of DC maturation, regarding the progression of atherosclerosis in ApoE-deficient mice. Materials and methods ApoE-deficient mice were fed for twelve weeks with a Western-type diet (n = 32) or normal chow (control group; n = 16). Animals receiving high-fat diet were treated with MCS-18 (500 μg/kg body weight, n = 16) or saline (n = 16) twice a week. After 12 weeks, animals were transcardially perfused and sacrificed. The percentage of mature DCs (CD3-/CD19-/CD14-/NK1.1-/CD11c+/MHCII+/CD83+/CD86+) and T cell subpopulations (CD4+/CD25+/Foxp3+, CD3/CD4/CD8) was analyzed in peripheral blood and in the spleen using flow cytometry. Plaque size was determined in the aortic root and the thoracoabdominal aorta using en-face staining. Immunohistochemical stainings served to detect inflammatory cells in the aortic root. Several cytokines and chemokines were determined in serum using multiplex assays. Results In splenic cells derived from saline-treated atherosclerotic mice an increased DC maturation, reflected by the upregulation of CD83 and CD86 expression, was observed. The enhanced expression of both maturation markers was absent in MCS-18 treated atherosclerotic mice. While the percentage of splenic Foxp3 expressing Treg was increased in animals receiving MCS-18 compared to saline-treated atherosclerotic mice, cytotoxic T cells were reduced in the spleen and in atherosclerotic lesions of the aortic root. Furthermore, proatherogenic cytokines (e.g. IL-6 and IFN-γ) and chemokines (e.g. MIP-1β) were decreased in serum of MCS-18-treated animals when compared to saline-treated atherosclerotic mice. Also plaque size in the aortic root and the thoracoabdominal aorta was significantly lower following administration of MCS-18. Conclusion This study provides for the first time evidence that MCS-18 is able to prevent the onset of atherosclerosis in ApoE-deficient mice. The observed anti-atherogenic effect is associated with the suppression of DC maturation and an inhibited migration and proliferation of cytotoxic T cells. [ABSTRACT FROM AUTHOR]

The physiological balance between pro- and anti-inflammatory processes is dysregulated in inflammatory bowel diseases (IBD) as in Crohn's disease and ulcerative colitis. Conventional therapy uses anti-inflammatory and immunosuppressive corticosteroids to treat acute-phase symptoms. However, low remission rate and strong side effects of these therapies are not satisfying. Thus, there is a high medical need for new therapeutic strategies. Soluble CD83, the extracellular domain of the transmembrane CD83 molecule, has been reported to have interesting therapeutic and immunosuppressive properties by suppressing dendritic cell (DC)-mediated T-cell activation and inducing tolerogenic DCs. However, the expression and function of CD83 in IBD is still unknown. Here, we show that CD83 expression is upregulated by different leukocyte populations in a chemical-induced murine colitis model. Furthermore, in this study the potential of sCD83 to modulate colitis using an experimental murine colitis model was investigated. Strikingly, sCD83 ameliorated the clinical disease symptoms, drastically reduced mortality, and strongly decreased inflammatory cytokine expression in mesenteric lymph nodes and colon. The infiltration of macrophages and granulocytes into colonic tissues was vigorously inhibited. Mechanistically, we could show that sCD83-induced expression of indolamine 2,3-dioxygenase is essential for its protective effects. [ABSTRACT FROM AUTHOR]

Abstract: The demand for human monocyte-derived dendritic cells (moDCs), as well as for primary human B and T lymphocytes for immunological research purposes has been increased in recent years. Classically, these monocytes are isolated from blood, leukapheresis products or buffy coats of healthy donors by plastic adherence of peripheral blood mononuclear cells (PBMCs), followed by stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, while lymphocytes are usually isolated from the non-adherent fraction (NAF) by magnetic cell sorting. However, donor-blood is a limited resource and not every blood bank offers leukapheresis products or buffy coats for laboratory use. Additionally, a leukapheresis is very expensive and also the generation/isolation of cells is time- and cost-intensive. To overcome some of these obstacles, we evaluated if low-cost leukoreduction system chambers (LRSCs), which arise after routine donor plateletpheresis procedures, and are usually discarded, would be an alternative and appropriate source of PBMCs to generate moDCs and to isolate lymphocytes. By analyzing the number and phenotype of immature and mature dendritic cells (DCs), as well as of B and T lymphocytes derived from LRSCs, we found all cells to be of high quantity and quality. Further investigations on DCs comprising transwell migration assays, allogeneic mixed lymphocyte reactions (MLR), cytokine secretion assays, and cytotoxic T cell induction assays revealed high migratory, as well as stimulatory capacity of these cells. In addition, DCs and T cells were efficiently electroporated with mRNA and showed characteristic cytokine production after co-culture, demonstrating LRSCs as an efficient, valid, and economic source for generation of moDCs and lymphocytes for research purposes. [Copyright &y& Elsevier]

Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25+ cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and T,.eg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25+ cell killing that may affect their clinical use and could be tested for novel indications. This trial was registered at www.clinical trials.gov, #NCT00056134. [ABSTRACT FROM AUTHOR]

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-β. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-β, 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. [ABSTRACT FROM AUTHOR]

Objective: To describe how a hydronephrosis can lead to a difficult differential diagnosis between endometriosis and retroperitoneal fibrosis. Design: Case report. Setting: Department of Obstetrics and Gynecology, Sacro Cuore Don Calabria General Hospital, Negrar, Verona, Italy. Patient(s): The history of a 34-year-old woman revealed the appearance of hydroureteronephrosis on the right side at the 35th week of pregnancy. She had an magnetic resonance imaging scan and was diagnosed with a spread retroperitoneal fibrosis. After 2 months, the patient reported the occurrence of pelvic pain, dyspareunia and dysmenorrhea. She was treated with corticosteroids and tamoxifen with no results. Intervention(s): Laparoscopic surgery. A complete retroperitoneal extirpation was done of an endometriotic nodule of the right broad ligament, near the right ureter (without stenosis). Main Outcome Measure(s): Reduction of pelvic pain. Result(s): She noticed an important decrease of pain. Conclusion(s): The cause of hydronephrosis could be a physiologic hydroureteronephrosis, which is the most common cause of dilatation of the urinary tract in pregnancy. The pain symptoms of the patients seemed to be linked to endometriosis and not to retroperitoneal fibrosis. Magnetic resonance imaging sometimes does not enable a correct diagnosis between these two pathologies. Fertile women with suspected fibrosis should undergo a diagnostic laparoscopy by an expert surgeon in retroperitoneal surgery. [Copyright &y& Elsevier]

Denileukin diftitox, also known as DAB389IL-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 a-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-&ggr; production of autoreactive splenic cells and a decreased presence of regulatory CD4+CD2S+ Foxp3+ T cells. In contrast, transfer of CD4+CD25+Foxp3+ T cells could correct the defect after denileukin diftitox treatment. Furthermore, whereas IFN-&ggr; 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. The Journal of Immunology, 2012, 189: 1173-1181. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Dendritic cells (DC) are the most potent antigen-presenting cells (APC) of the immune system and are specialized to activate T as well as B cell-dependent immune responses. Mature DC are characterized by expression of CD83, a surface molecule that has been postulated to be required for efficient DC activity. Here we show that Leptomycin B (LMB), a highly specific inhibitor of the nuclear export receptor CRM1, abrogates the ability of DC to stimulate T cells in an allogeneic mixed lymphocyte reaction. Interestingly, this effect correlates with down-regulation of CD83, CD80 and CD86 surface expression during DC maturation, whereas other investigated DC surface molecules, such as MHC class I and II molecules are not significantly affected. Analysis of RNA distribution reveals that particularly the stimulated expression of CD83 depended on a functional CRM1 export receptor. Taken together, the presented data show a critical involvement of the CRM1 transport receptor in DC maturation, most likely by enabling efficient nucleo-cytoplasmic translocation of specific mRNAs. Thus, interference with this pathway may provide new strategies to modulate DC function and, subsequently, DC-mediated immune responses. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Mature dendritic cells (mDCs) are the most potent antigen presenting cells within the human immune system known today. However, several viruses, including herpes simplex virus type 1 (HSV-1) have developed numerous immune escape mechanisms, such as the avoidance of peptide presentation through the major histocompatibility complex (MHC) class I to CD8+ cytotoxic T-cells. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome, a multicatalytic protease complex. Upon exposure to IFN-γ, the constitutive proteasome is partially replaced by the immunoproteasome, which contains the IFN-γ-inducible subunits LMP2, MECL1 and LMP7. In this study, we report the downregulation of LMP7 on mRNA level in HSV-1 infected mDCs. Interestingly, this reduction was not vhs-mediated since using a virus strain lacking the vhs gene we obtained similar results. However, on protein level, LMP7-expression was not affected, which is probably due the high stability of the LMP7 protein. Also the incorporation of LMP7 into the immunoproteasome was not affected by HSV-1. However, for the in vivo situation, in which DC reside for a prolonged time period in peripheral tissues, the reduced LMP7-mRNA level could be of biological importance, since the virus could escape/hide from immune system of the host and establish latency processes. [Copyright &y& Elsevier]

Abstract: Dendritic cells are the sentinels of the immune system and as such represent the first-line of defense against incoming pathogens. Upon encounter with harmful antigens, these antigen-presenting cells start to mature and migrate towards the draining lymph nodes to display the antigen to T-lymphocytes, thereby eliciting the immune response of the host. Viruses, including human herpesvirus type I (HSV-1), seek to avoid such immune reactions. Therefore, they developed an arsenal of immune evasion strategies, some of which have been described earlier by our group and others. The secretion of tumor necrosis factor (TNF) represents a typical defense line of the host and it has been shown that this cytokine contributes to the inhibition of viral replication and augments the proliferation of cytotoxic T-lymphocytes. Here we report, that upon infection of mature dendritic cells, HSV-1 very strongly induces the expression of the AU-rich elements (ARE)-binding protein tristetraprolin (TTP), an mRNA-destabilizing protein. One of the best described targets of TTP is the TNF mRNA. This induction is dependent on the phosphorylation of both signal transducer and activator of transcription (STAT1) and p38 in a collaborative manner. By repressing this phosphorylation with specific inhibitors, we were able to reduce TTP mRNA levels. At the same time TNF mRNA levels were increased, suggesting that TNF mRNA is indeed a target of TTP in this setting. In summary, these data underline that HSV-1 induces TTP transcription in order to reduce TNF levels generated by infected mature dendritic cell. [Copyright &y& Elsevier]

Abstract: Soluble CD83 (sCD83), a potent immunosuppressive agent, circulates at elevated levels in some chronic lymphocytic leukemia (CLL) patients. We report that CLL patients with elevated plasma sCD83 levels had significantly shorter (P =0.038) treatment free survival. Culture of CLL cells with solid phase CD83 mAb+IL-4 significantly increases sCD83 release (23–117-fold, P =0.013) and ligation of normal donor PBMC with solid phase CD83 mAb alone induces similar significant increases in sCD83 release (P =0.003). RT-PCR analysis detected the presence of a transcript for sCD83 in 2/3 CLL samples. These results suggest sCD83 release may play a regulatory role in CLL progression. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Selective gene silencing by RNA interference (RNAi) has been shown to be an efficient method for the targeted manipulation of cellular functions. In this study we describe for the first time electroporation as a suitable and efficient method for the delivery of small interfering RNA (siRNA) into murine bone marrow-derived dendritic cells (BM-DC). Using a fluorescein-labeled non-silencing siRNA duplex, we established an electroporation protocol yielding routinely >90% positive cells. We investigated the effects of siRNA electroporation on BM-DC viability, phenotype and ability to induce allogeneic T cell proliferation. Finally, using siRNAs directed against MAPK1 and the transcription factor HIF-1α we were able to demonstrate an efficient knock down of cellular mRNA- and protein level in electroporated BM-DC. Furthermore, knocking down the transcription factor HIF-1α impeded hypoxic induction of HIF-1α target genes. We therefore propose siRNA electroporation into murine BM-DC as an efficient method to manipulate BM-DC function without the use of chemical transfection reagents. This new approach is superior to lipofection regarding detrimental effects of lipid-based transfection agents on BM-DC immunobiology. [Copyright &y& Elsevier]

Abstract: Here we report for the first time that MCS-18, a novel natural product isolated from Helleborus purpurascens, is able to inhibit the expression of typical molecules of mature dendritic cells (DC) such as CD80, CD86, and especially of CD83 subsequently leading to a clear and dose-dependent inhibition of the DC-mediated T-cell stimulation. Furthermore, MCS-18 impeded the formation of the typical DC/T-cell clusters, which are essential to induce potent immune responses. Interestingly, MCS-18 also inhibited CCR7 expression on DC which subsequently lead to a dose-dependent block of the CCL19-mediated DC migration. MCS-18 not only inhibited the DC-mediated T-cell stimulation but also the anti-CD3/anti-CD28-mediated T-cell stimulation. Strikingly, MCS-18 also strongly reduced the paralysis associated with the experimental autoimmune encephalomyelitis (EAE), which is a murine model for human multiple sclerosis, in a prophylactic as well as in a “real” therapeutic setting. Even when the EAE was induced for a second time, the MCS-18-treated animals were still protected, suggesting that MCS-18 induces a long-lasting suppressive effect. In addition, and very important for the potential practical application in humans, MCS-18 was also active when administered orally. MCS-18 treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In conclusion, using in vitro as well in vivo assays we were able to show that MCS-18 exerts a strong immunosuppressive activity with remarkable potential for the therapy of diseases characterized by a pathologically over-activated immune system. [Copyright &y& Elsevier]

Abstract: Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) of the immune system. Their migration to secondary lymphoid tissues is a crucial step for the priming of T cells and ultimately for the initiation of adaptive immune responses. Therefore, DCs are potential targets for immune evasion strategies of pathogens. The migration of DCs to the T cell areas of lymph nodes is guided by a gradient of chemokines, CCL19 and CCL21, which are constitutively expressed there. CCR7, the receptor for these chemokines, is expressed on activated DCs, enabling their homing to the lymph nodes. However, CCR7 expression alone is not sufficient for efficient migration. Prostaglandin E2 (PGE2) is a mandatory factor for CCR7-mediated migration of DCs and exerts its effects via prostaglandin E2 receptor 2 (EP2) and prostaglandin E2 receptor 4 (EP4). In this study, we investigated the effect of herpes simplex virus type 1 (HSV-1) infection of mature monocyte-derived dendritic cells (MODCs) on the EP2 and EP4 receptor expression. Affymetrix analyses and real-time polymerase chain reaction (PCR) demonstrated a dramatic down-regulation of the expression of those receptors. Additional real-time PCR and migration assays with a Δvhs mutant virus lacking the virion host shutoff (vhs) gene implicate a vhs independent mechanism. Therefore, our results suggest a novel immune evasion strategy for HSV-1. [Copyright &y& Elsevier]

Abstract: A soluble form of CD83 (“sCD83”) has been shown to block DC-mediated T cell stimulation in vitro and an immunosuppressive role has also been shown in vivo using an experimental-autoimmune-encephalomyelits (EAE) model. Using recombinant mutational analyses, recently, we could show that sCD83 forms a homo-dimer, whereby four cysteines are involved in the intra-molecular disulfide bonds and the fifth cysteine is responsible for the inter-molecular bridging of the two molecules. Further studies revealed that the two CD83-isoforms, i.e. the dimer and the monomer, have a similar inhibitory capacity when tested in vitro. Here we show that the biological (in vivo) half-life of the two sCD83 isoforms is comparable and was between 2 and 3h. In addition, using the EAE-model, we were able to show that a monomeric-mutant isoform of soluble CD83 has a similar inhibitory activity in vivo when compared with a dimeric-wildtype isoform. [Copyright &y& Elsevier]

The aryl hydrocarbon receptor (AhR) is an environmental sensor and ligand-activated transcription factor that is critically involved in the regulation of inflammatory responses and the induction of tolerance by modulating immune cells. As dendritic cells (DCs) express high AhR levels, they are efficient to induce immunomodulatory effects after being exposed to AhR-activating compounds derived from the environment or diet. To gain new insights into the molecular targets following AhR-activation in human monocyte-derived (mo)DCs, we investigated whether the natural AhR ligand quercetin or the synthetic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) modulates the function of human moDCs regarding their capability to prime naïve T cells or to migrate. As only quercetin, but not TCDD, impaired T cell activation and migration of LPS-matured DCs (LPS-DCs), we analyzed the mode of action of quercetin on moDCs in more detail. Here, we found a specific down-regulation of the immunomodulatory molecule CD83 through the direct binding of the activated AhR to the CD83 promoter. Furthermore, treatment of LPS-DCs with quercetin resulted in a reduced production of the pro-inflammatory cytokine IL-12p70 and in an increased expression of the immunoregulatory molecules disabled adaptor protein (Dab) 2, immunoglobulin-like transcript (ILT)-3, ILT4, ILT5 as well as ectonucleotidases CD39 and CD73, thereby inducing a tolerogenic phenotype in quercetin-treated maturing DCs. Overall, these data demonstrate that quercetin represents a potent immunomodulatory agent to alter human DC phenotype and function, shifting the immune balance from inflammation to resolution. [ABSTRACT FROM AUTHOR]

Mature dendritic cells (DCs) are the most potent antigen-presenting cells within the human immune system. However, Herpes simplex virus type 1 (HSV-1) is able to interfere with DC biology and to establish latency in infected individuals. In this study, we provide new insights into the mechanism by which HSV-1 disarms DCs by the manipulation of CD83, a functionally important molecule for DC activation. Fluorescence-activated cell sorter (FACS) analyses revealed a rapid downmodulation of CD83 surface expression within 6 to 8 h after HSV-1 infection, in a manner strictly dependent on viral gene expression. Soluble CD83 enzyme-linked immunosorbent assays, together with Western blot analysis, demonstrated that CD83 rapidly disappears from the cell surface after contact with HSV-1 by a mechanism that involves protein degradation rather than shedding of CD83 from the cell surface into the medium. Infection experiments with an ICP0 deletion mutant demonstrated an important role for this viral immediate-early protein during CD83 degradation, since this particular mutant strain leads to strongly reduced CD83 degradation. This hypothesis was further strengthened by cotransfection of plasmids expressing CD83 and ICP0 into 293T cells, which led to significantly reduced accumulation of CD83. In strong contrast, transfection of plasmids expressing CD83 and a mutant ICP0 defective in its RING finger-mediated E3 ubiquitin ligase function did not reduce CD83 expression. Inhibition of the proteasome, the cellular protein degradation machinery, almost completely restored CD83 surface expression during HSV-1 infection, indicating that proteasome-mediated degradation and HSV-1 ICP0 play crucial roles in this novel viral immune escape mechanism. [ABSTRACT FROM AUTHOR]

Objectives: Metabolic changes are crucially involved in osteoclast development and may contribute to bone degradation in rheumatoid arthritis (RA). The enzyme aconitate decarboxylase 1 (Acod1) is known to link the cellular function of monocyte-derived macrophages to their metabolic status. As osteoclasts derive from the monocyte lineage, we hypothesised a role for Acod1 and its metabolite itaconate in osteoclast differentiation and arthritis-associated bone loss., Methods: Itaconate levels were measured in human peripheral blood mononuclear cells (PBMCs) of patients with RA and healthy controls by mass spectrometry. Human and murine osteoclasts were treated with the itaconate derivative 4-octyl-itaconate (4-OI) in vitro. We examined the impact of Acod1-deficiency and 4-OI treatment on bone erosion in mice using K/BxN serum-induced arthritis and human TNF transgenic (hTNFtg) mice. SCENITH and extracellular flux analyses were used to evaluate the metabolic activity of osteoclasts and osteoclast progenitors. Acod1-dependent and itaconate-dependent changes in the osteoclast transcriptome were identified by RNA sequencing. CRISPR/Cas9 gene editing was used to investigate the role of hypoxia-inducible factor (Hif)-1α in Acod1-mediated regulation of osteoclast development., Results: Itaconate levels in PBMCs from patients with RA were inversely correlated with disease activity. Acod1-deficient mice exhibited increased osteoclast numbers and bone erosion in experimental arthritis while 4-OI treatment alleviated inflammatory bone loss in vivo and inhibited human and murine osteoclast differentiation in vitro. Mechanistically, Acod1 suppressed osteoclast differentiation by inhibiting succinate dehydrogenase-dependent production of reactive oxygen species and Hif1α-mediated induction of aerobic glycolysis., Conclusion: Acod1 and itaconate are crucial regulators of osteoclast differentiation and bone loss in inflammatory arthritis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Alterations in macrophage (Mφ) polarization, function, and metabolic signature can foster development of chronic diseases, such as autoimmunity or fibrotic tissue remodeling. Thus, identification of novel therapeutic agents that modulate human Mφ biology is crucial for treatment of such conditions. Herein, we demonstrate that the soluble CD83 (sCD83) protein induces pro-resolving features in human monocyte-derived Mφ biology. We show that sCD83 strikingly increases the expression of inhibitory molecules including ILT-2 (immunoglobulin-like transcript 2), ILT-4, ILT-5, and CD163, whereas activation markers, such as MHC-II and MSR-1, were significantly downregulated. This goes along with a decreased capacity to stimulate alloreactive T cells in mixed lymphocyte reaction (MLR) assays. Bulk RNA sequencing and pathway analyses revealed that sCD83 downregulates pathways associated with pro-inflammatory, classically activated Mφ (CAM) differentiation including HIF-1A, IL-6, and cytokine storm, whereas pathways related to alternative Mφ activation and liver X receptor were significantly induced. By using the LXR pathway antagonist GSK2033, we show that transcription of specific genes (e.g., PPARG , ABCA1 , ABCG1 , CD36 ) induced by sCD83 is dependent on LXR activation. In summary, we herein reveal for the first time mechanistic insights into the modulation of human Mφ biology by sCD83, which is a further crucial preclinical study for the establishment of sCD83 as a new therapeutical agent to treat inflammatory conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Peckert-Maier, Wild, Sprißler, Fuchs, Beck, Auger, Sinner, Strack, Mühl-Zürbes, Ramadan, Kunz, Krönke, Stich, Steinkasserer and Royzman.)

Microglial activation during neuroinflammation is crucial for coordinating the immune response against neuronal tissue, and the initial response of microglia determines the severity of neuro-inflammatory diseases. The CD83 molecule has been recently shown to modulate the activation status of dendritic cells and macrophages. Although the expression of CD83 is associated with early microglia activation in various disease settings, its functional relevance for microglial biology has been elusive. Here, we describe a thorough assessment of CD83 regulation in microglia and show that CD83 expression in murine microglia is not only associated with cellular activation but also with pro-resolving functions. Using single-cell RNA-sequencing, we reveal that conditional deletion of CD83 results in an over-activated state during neuroinflammation in the experimental autoimmune encephalomyelitis model. Subsequently, CD83-deficient microglia recruit more pathogenic immune cells to the central nervous system, deteriorating resolving mechanisms and exacerbating the disease. Thus, CD83 in murine microglia orchestrates cellular activation and, consequently, also the resolution of neuroinflammation., (© 2023. The Author(s).)

Rationale: Sepsis, a global health burden, is often complicated by viral infections leading to increased long-term morbidity and mortality. Interleukin-3 (IL-3) has been identified as an important mediator amplifying acute inflammation in sepsis; however, its function in the host response to viral infections during sepsis remains elusive., Objectives: To investigate the role of IL-3 during viral pneumonia in sepsis., Methods: We included septic patients from two different cohorts and used in vitro and in vivo assays. The obtained data were substantiated using a second model (SARS-CoV-2 infections)., Measurements and Main Results: Low plasma IL-3 levels were associated with increased herpes simplex virus (HSV) airway infections in septic patients, resulting in reduced overall survival. Likewise, Il-3 -deficient septic mice were more susceptible to pulmonary HSV-1 infection and exhibited higher pulmonary inflammation than control mice. Mechanistically, IL-3 increases innate antiviral immunity by promoting the recruitment of circulating plasmacytoid dendritic cells (pDCs) into the airways and by enhancing pDC-mediated T cell activation upon viral stimulation. Interestingly, the ability of IL-3 to improve adaptive immunity was confirmed in patients with SARS-CoV-2 infections., Conclusion: Our study identifies IL-3 as a predictive disease marker for viral reactivation in sepsis and reveals that IL-3 improves antiviral immunity by enhancing the recruitment and the function of pDCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bénard, Hansen, Uhle, Klösch, Czubayko, Mittelstädt, Jacobsen, David, Podolska, Anthuber, Swierzy, Schaack, Mühl-Zürbes, Steinkasserer, Weyand, Weigand, Brenner, Krautz, Grützmann and Weber.)

Excessive macrophage (Mφ) activation results in chronic inflammatory responses or autoimmune diseases. Therefore, identification of novel immune checkpoints on Mφ, which contribute to resolution of inflammation, is crucial for the development of new therapeutic agents. Herein, we identify CD83 as a marker for IL-4 stimulated pro-resolving alternatively activated Mφ (AAM). Using a conditional KO mouse (cKO), we show that CD83 is important for the phenotype and function of pro-resolving Mφ. CD83-deletion in IL-4 stimulated Mφ results in decreased levels of inhibitory receptors, such as CD200R and MSR-1, which correlates with a reduced phagocytic capacity. In addition, CD83-deficient Mφ upon IL-4 stimulation, show an altered STAT-6 phosphorylation pattern, which is characterized by reduced pSTAT-6 levels and expression of the target gene Gata3 . Concomitantly, functional studies in IL-4 stimulated CD83 KO Mφ reveal an increased production of pro-inflammatory mediators, such as TNF-α, IL-6, CXCL1 and G-CSF. Furthermore, we show that CD83-deficient Mφ have enhanced capacities to stimulate the proliferation of allo-reactive T cells, which was accompanied by reduced frequencies of Tregs. In addition, we show that CD83 expressed by Mφ is important to limit the inflammatory phase using a full-thickness excision wound healing model, since inflammatory transcripts (e.g. Cxcl1, Il6 ) were increased, whilst resolving transcripts (e.g. Ym1, Cd200r, Msr-1 ) were decreased in wounds at day 3 after wound infliction, which reflects the CD83 resolving function on Mφ also in vivo . Consequently, this enhanced inflammatory milieu led to an altered tissue reconstitution after wound infliction. Thus, our data provide evidence that CD83 acts as a gatekeeper for the phenotype and function of pro-resolving Mφ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Peckert-Maier, Langguth, Strack, Stich, Mühl-Zürbes, Kuhnt, Drassner, Zinser, Wrage, Mattner, Steinkasserer, Royzman and Wild.)

To facilitate the recovery process of chronic and hard-to-heal wounds novel pro-resolving treatment options are urgently needed. We investigated the pro-regenerative properties of soluble CD83 (sCD83) on cutaneous wound healing, where sCD83 accelerated wound healing not only after systemic but also after topical application, which is of high therapeutic interest. Cytokine profile analyses revealed an initial upregulation of inflammatory mediators such as TNFα and IL-1β, followed by a switch towards pro-resolving factors, including YM-1 and IL-10, both expressed by tissue repair macrophages. These cells are known to mediate resolution of inflammation and stimulate wound healing processes by secretion of growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), which promote vascularization as well as fibroblast and keratinocyte differentiation. In conclusion, we have found strong wound healing capacities of sCD83 beyond the previously described role in transplantation and autoimmunity. This makes sCD83 a promising candidate for the treatment of chronic- and hard-to-heal wounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Royzman, Peckert-Maier, Stich, König, Wild, Tauchi, Ostalecki, Kiesewetter, Seyferth, Lee, Eming, Fuchs, Kunz, Stürmer, Peters, Berking, Zinser and Steinkasserer.)